TM
TM
TM
guava
Benchtop Flow Cytometry Kits and Reagents
WHAT’S INSIDE
Cell Health• Cell Counting & Viability• Cell Cycle• Mitochondrial Analysis• Apoptosis
Cell Signaling• MAPK Pathway• EGFR Pathway• PI3/Akt/mTOR Pathway• Jak/STAT Pathway• DNA Damage Pathway• Chemokine
Stem Cells• Embryonic Stem Cell
(Human/Mouse)• Neural Stem Cell (Rodent)
Immunology• Regulation T-Cell• Phenotype Markers
Milli-Mark™Conjugated Antibodies
Components of the guava® Flow Cytometry Solution• Instruments: easyCyte™
Flow Cytometers • Software• Kits & Reagents
FROM CORE LAB TO YOUR LAB—GUAVA® INTEGRATED FLOW CYTOMETRY SOLUTIONSFlow cytometry is an essential tool for in-depth cell analysis. With the capacity to simultaneously measure multiple parameters on hundreds of individual cells per second, flow cytometry offers greater speed, precision, and detail than most other cell analysis methods available to scientists today. Integrated products from Millipore will help streamline your workflow. Our complete benchtop flow cytometry solution includes our instruments, assays, and software—as well as the cell isolation and culture tools to prepare your samples. With a flow cytometry solution right in your lab, you’ll experience superior performance, higher quality data and faster progress from hypothesis to results.
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Antibody Selectionand Assay DesignThe antibodies used in each kit are carefully selected by reviewing many publications.
Ab Component ValidationAntibodies are screened primarily by Western blot to determine specificity, then validated with secondary antibody for flow cytometry use.
Ab Conjugation and TestingAntibodies are conju-gated to fluorophores that will ensure less fluorescence spectrum overlap.
Multiplexing, Stability, Performance ClaimsThe antibody conjugates are optimized to provide the best signal-to-noise ratio when multiplexing.
FlowCellect Kits and Milli-MarkConjugated AntibodiesMany researchers invest time optimizing and validating
antibodies for flow cytometry, only to discover that these
antibodies do not perform well when multiplexed together,
because of interference from the matrix or from other
antibodies.
Millipore’s FlowCellect kits and Milli-Mark conjugated
primary antibodies are fully optimized for fast, easy, and
accurate multiparametric flow cytometry. We’ve taken the
guesswork out of assay development so you can focus
on your results. We optimize and validate every antibody,
making sure they work together well. All you need are cells
and a research question; our assay kits will do the rest, and
you’ll have data before your cells are ready to split again.
FlowCellect Kits FlowCellect kits are Millipore’s proprietary multiparameter
flow cytometry kits for the analysis of cellular events
and/or cell phenotypes. Each kit has unique combination
of directly conjugated antibodies, and/or fluorescent
dyes and protein reporters to monitor changes in protein
expression and posttranslational modification. The kits also
contain complete buffer sets, protocols and pre-defined
gate settings. They are fully optimized for “plug-and-play”
cellular analysis on guava and other flow cytometers.
Advantages• Multiplexing capabilities
• Easy to use, with fewer incubation and wash steps
• Fully validated and concentration-optimized,
guaranteed to work in flow cytometry
Using a four-step validation process to develop our
FlowCellect kits, we’ve eliminated the need to design your
experiment or optimize antibodies and buffers:
Milli-Mark Conjugated Antibodies Milli-Mark antibodies are directly conjugated primary
antibodies that are validated for flow cytometry in
addition to traditional applications like Western blotting
and immunocytochemistry (see figure 1b for an example
of antibody validation). Because of their extensive cross-
platform validation, Milli-Mark antibodies are valuable,
convenient building blocks with which you can configure
your own assays.
FlowCellect Kit Development Steps
250
130
100
70
55
35
27
15
10
10
15
27
35
55
70
100
130
250
44kDa
OCT-4 antibody
SSEA-4 antibody
70kDa
Fig. 1. 1a. Mouse embryonic stem cells and fibroblasts labeled with Oct-4 (green), SSEA-1 (red), and Hoechst nuclear stain (blue). 1b. Human embryonic stem cell lysate.
Kits and Reagents
2
Knowing the performance profile of your cells prior to
running your bioassay can mean the difference between
valid assay results and wasted reagents, lost time and
discarded data.
Monitoring key indicators of cell health, such as
the apoptotic fraction and stage of apoptosis, viability,
cell cycle, cell counts, transfection efficiency, and target
expression levels, helps establish uniform standards of
cellular performance across long-term research studies.
These standards can be applied to a wide range of
bioassays. Whether you are establishing screen/no screen
criteria for high throughput screening, monitoring and
optimizing bioreactor conditions, or eliminating sources of
assay variability, consistent monitoring of your cell model
improves your bioassay performance and productivity.
CELL COUNTING AND VIABILITYCell counting and viability assessments can be used in a
variety of applications, such as cytotoxicity studies, PBMC
counting and rapid apoptosis assessment.
guava ViaCount® Assay KitThe guava ViaCount assay is fast becoming the new
standard for viability and cell counting. In this simple
no-wash, mix-and-read-assay, you can accurately obtain
absolute total cell counts, perform viability assessments
and determine apoptotic percentages—all from tiny
samples. You’ll enjoy several advantages over traditional
methods, including greater accuracy, reproducibility and
speed.
Cell Signaling• Chemokine
Viability Stain
Nuc
leat
ed C
ells
Live
Apoptotic
Dead
Annexin V
Via
bilit
y Sta
in
Live
Apoptotic
Dead
FL3
-Hei
ght
FL2-Height
2
FL2
-Hei
ght
2
Fig. 2. The blue population of cells show a significant amount of annexin V staining (right plot), indicating that intermediate levels of staining with the viability dye correlates with apoptosis.
ORDERING INFORMATIONDescription Qty/Pk Catalogue No.
Guava ViaCount Reagent Kit 100 Tests 4500-0040
Guava ViaCount Reagent Kit 600 Tests 4500-0041
Guava ViaCount Flex Reagent Kit for challenging samples
100 Tests 4500-0110
Guava ViaCount Flex Reagent Kit 500 Tests 4700-0060
Guava ViaCount Cell Dispersal Reagent
100 Tests 4700-0050
Cell Health
AdvantagesAssay:• Simple no-wash, mix-and-read procedure
• Counts up to 10 times faster than manual methods
• More reproducible than traditional testsSamples:• Uses small samples in tubes or a 96-well plate
• Handles low-density and small-volume cell samples
• Works with adherent or suspended cells and mammalian and insect cells
Discover the power of flow cytometry for multiparametric cell health analysis.
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Discover the power of flow cytometry for multiparametric cell cycle research.
CELL CYCLECell cycle phase distributions can be used to assess
cell health and proliferation and studying the potential
mechanism of antineoplastic agents. For example,
measuring the population of S phase cells can reflect the
amount of newly synthesized DNA. Also, distinguishing cells
in G2 from M phase cells can help identify cells undergoing
mitosis.
Flow cytometry analysis of cell DNA content has been
one of the best and most popular tools for researchers
to be widely used for the estimation of cell cycle phase
distribution. However, the limitation of single-marker
analysis, such as a DNA dye only, is that cells within
each phase cannot accurately be determined without
mathematical interpolation using analysis software.
The cell cycle can be divided into two distinct stages.
The first stage is interphase which consists of the G1, S,
and G2 phases, in which cells are active, growing, and DNA
is being replicated. The second is M-phase, also known as
the “mitotic phase”, in which cell division takes place.
FlowCellect Cell Cycle Kits Millipore has developed and optimized two bivariate cell
cycle analysis kits using phase-specific antibodies in
addition to a DNA dye. Bivariate analysis will not only reveal
the cell distribution within a particular phase of cell cycle,
but can also enable the researcher to elucidate mechanisms
of cell cycle regulation, without sophisticated software
modules or algorithms.
Cell Growth
Cell Prepares for Division
DNA Synthesis
Mitosis
Cells that cease dividing
I N T E R P H A S E
Cytokinesi s
FEATURED PRODUCT
FlowCellect Bivariate Cell Cycle Kit for DNA Replication Analysis Investigate DNA replication in the S phase with high
accuracy and confidence. The kit includes a directly
conjugated Anti-BrdU Alexa Fluor® 488 antibody plus
a DNA dye (propidium iodide). BrdU incorporation is a
widely accepted method of measuring DNA replication
and kinetics of cell cycle progression. The percentage of
BrdU labeled cells is a reliable estimate of the S phase
compartment, and labeled cells can then be followed
through the cell cycle.
Fig. 3. Detection of DNA replication by analysis of S phase cells. Bivariate flow cytometric analysis us-ing BrdU Alexa Fluor 488 conjugate can distinguish S phase cells with great accuracy, not only based on their difference in DNA content from G1 or G2/M cells but also as having incorporated BrdU.
G=24% (-BrdU, 1X DNA content)
S=72% (↑BrdU, 1-2X DNA content) G2/M=4% (-Brdu, 2X DNA content)
Advantages• No specific cell cycle analysis software required
• Quantitative measurements of percentage of cells
within each cell subpopulation
• Minimal assay development needed
• Includes all optimized flow cytometry antibodies
and buffers
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FlowCellect Bivariate Cell Cycle Kit for G2/M Analysis (Application)
Fig. 5. Nocodazole treatment increases percentage of cells in M phase. Cell were either treated with 100 µm Nocodazole (test sample) or left untreated (control) overnight at 37 ºC. By plotting the phosohorylation of histone H3 at Ser10 (y axis) versus DNA content (x axis), an increase in the proportion of G2/M cells was observed indicating that mitotic cells have accumu-lated after treatment. Apporomately 2% of cells reside in M phase under normal conditions in Jurkat cells, but when treated cell population increased 18%.
guava Cell Cycle AssayThis kit uses the nuclear DNA stain, propidum iodide (PI), to
measure cell cycle. Resting cells (G0/G1) contain two copies
of each chromosome. Cycling cells synthesize chromosomal
DNA (S phase), which results in increased fluorescence
intensity. When all chromosomal DNA has doubled (G2/M
phase), cells fluoresce with twice the intensity of the initial
population.
ORDERING INFORMATION FlowCellect Kits(using phase-specific antibodies plus a DNA dye)
Description Qty/Pk Catalogue No.
Bivariate Cell Cycle Kit for DNA Replication Analysis Anti-BrdU / Propidium Iodide Solution
25 tests FCCH025102
Bivariate Cell Cycle Kit for G2/M Analysis Anti-phospho-Histone H3(Ser10) / Propidium Iodide Solution
25 tests FCCH025103
FlowCellect Cell Cycle Check Point ATM DNA Damage Kit
25 tests FCCH025143
Flow Cellect Cell Cycle Check Point H2A.X DNA Damage Kit
25 tests FCCH025142
guava Cell Cycle Reagent Propidium Iodide Solution
100 tests 4500-0220
Cell Cycle Phases:G1 = 57%S = 19%G2 = 15%M = 3%
FEATURED PRODUCT
FlowCellect Bivariate Cell Cycle Kit for G2/M AnalysisInvestigate the G2/M phase transition with high
accuracy and confidence. The phosphorylation of
histone H3 at Ser10 correlates with the G2 to M
phase transition and is a prerequisite for chromatin
condensation at mitosis. At the end of mitosis,
histone H3 is rapidly dephosphorylated and remains
unphosphorylated throughout the remainder of
interphase. Therefore, phospho-histone H3 (Ser10) is
a reliable, specific marker of M-phase cells.
Fig. 4. Discrimination between G2 and M phase cells by measuring the phosphorylation of histone H3 on Ser10. Histone H3 is constitutively phosphorylated at Ser10 during metaphase.
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Mitochondrial Signaling and ApoptosisExtrinsic pathway signal
Smac/DIABLO
OMI/HTRA2
Pro-Caspase 8Caspase 8
Caspase 8
Caspase 3Caspase 3
Casp 9
Apoptosome
ER stress,DNA damageOxidants
Pro-Caspase 9
APAF-1
EndoG
AIF
DNA fragmentationchromatin condensation
Bidt-Bid
t-Bid
Type II
Bax, Bak,
Type I
IAPIAP
Cyt c
Cyt c
Adapted from Bayir and Kagan Critical Care 2008 12:206.
MITOCHONDRIAL ANALYSISMitochondria are critical cellular organelles that produce
90% of cellular energy, control cell survival by regulating
apoptosis, and produce reactive oxygen species (ROS).
Mitochondrial superoxide generation results in of oxidative
stress, damage and cell death by apoptosis or to a cellular
energetic decline. Therefore, mitochondrial dysfunction
caused by disease or compound treatment has dire
consequences that can result in apoptosis, necrosis/cell
death, or caspase-independent cell death.
Monitoring impact on mitochondria and related cell
health markers is an important part of drug screening
programs, pathway mapping, and apoptosis and disease
research.
Flow cytometry detects multiple markers simultaneously
at various stages of apoptosis, making it a powerful
technique for studying pathways governing cell health and
cell death.
Discover the power of flow cytometry for multiparametric mitochondrial analysis.
FlowCellect Mitochondrial Kits These kits harness the power of flow cytometry to assess
changes in mitochondrial membrane potential, apoptosis
as measured by Annexin V binding, mitochondrial oxidative
stress, and cell death, using only minimal cellular samples.
The kits may be used with most dual laser flow cytometry
systems equipped with a 488 nm and a 644 nm laser.
AdvantagesAssay:• Multiplex detection with no optimization required
• Highly reproducible
• Minimal assay development needed
• All optimized flow cytometry antibodies and buffers
included
• Enables novice users to perform complex analysis
Samples:
• Designed to run 100 samples of human cells
FlowCellect MitoDamage KitKit Component Laser Buffer Pack
MitoSense Red Dye Red
10X Assay Buffer HSCAnnexin CF 488 Blue
7-AAD Blue
Simultaneously measures 3 important cell health
parameters:
• Change in mitochondrial potential (considered an early
hallmark of apoptosis or cell stress)
• Phospatidylserine translocation to the surface of early
apoptotic cells (measured by Annexin V binding)
• Plasma membrane permeabilization or cell death
(measured by 7-AAD)
Fig.6. Dot plots depicting Jurkat cells stained using the MitoDamage kit. Jurkat cells uninduced (column A), induced to apoptosis with 2 µM staurosporine (column B) or with 50 µM CCCP (column C), then stained using the MitoDamage kit. Plots show the percentage of positive cells for:
1st row: Apoptosis (Annexin V binding) and mitochondrial membrane potential change 2nd row: Cell death and mitochondrial membrane potential change 3rd row: Apoptosis and cell death. Data reports that 2 µM staurosporine induces apoptosis in Jurkat cells, and that 50 µM CCCP depolarizes the mitochondrial
membrane, but neither condition is sufficient for cell membrane permeabilization and death.
The FlowCellect MitoDamage kit can thus distinguish
multiple populations:
1. Healthy cells with intact mitochondrial membrane
2. Stressed cells with dissipated membrane potential
without Annexin V or 7-AAD staining
3. Early apoptotic cells with dissipated membrane
potential and Annexin V binding
4. Late apoptotic cells or dead cells with dissipated
membrane potentials
Mit
oSen
se R
ed
Annexin V, CF488A
Mit
oSen
se R
ed7-A
AD
Annexin V, CF488A
Red
2 F
luor
esec
ence
(R
D2
-HLo
g)
Green Fluorescence (GRN-HLog)
94.4%
0.75% 3.7%
Uninduced
100 101 102 103 104100
101
102
103
104
1.1%
Red
2 F
luor
esce
nce
(RD
2-H
Log)
Red Fluorescence (RED-HLog)
95.2%
3.2% 1.3%100 101 102 103 104
100
101
102
103
104
0.3%
Red
2 F
luor
esce
nce
(RD
2-H
Log)
Green Fluorescence (GRN-HLog)
0.16%
95.2% 3.2%100 101 102 103 104
100
101
102
103
104
1.4%
Red
2 F
luor
esce
nce
(RD
2-H
Log)
Green Fluorescence (GRN-HLog)
0.06%
70.5% 28.2%100 101 102 103 104
100
101
102
103
104
1.2%
Red
2 F
luor
esce
nce
(RD
2-H
Log)
Green Fluorescence (GRN-HLog)
0.10%
93.9% 4.8%100 101 102 103 104
100
101
102
103
104
1.2%
Red
2 F
luor
esce
nce
(RD
2-H
Log)
Red Fluorescence (RED-HLog)
0.26%
98.4% 1.3%100 101 102 103 104
100
101
102
103
104
0.02%
Red
2 F
luor
esec
ence
(R
D2
-HLo
g)
Green Fluorescence (GRN-HLog)
54.7%
14.6% 27.0%100 101 102 103 104
100
101
102
103
104
3.7%
Red
2 F
luor
esec
ence
(R
D2
-HLo
g)
Green Fluorescence (GRN-HLog)
0.20%
93.2% 6.6%
50 µM CCCP
100 101 102 103 104100
101
102
103
104
0.04%
100 101 102 103 104
101
102
103
104
Depolarized Cells
Dead Cells
Live Cells
58.1% 0.08%
41.0% 0.8%
Red
2 F
luor
esce
nce
(RD
2-H
Log)
Red Fluoresecence (RED-HLog)
100
2 µM Staurosporine
7-AAD
FEATURED PRODUCT
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7
FlowCellect MitoPotential Red KitSimultaneous analysis of mitochondrial membrane potential
along with cell death provides key information for drug
discovery, cancer and toxicology studies as well as disease-
induced apoptosis. This kit uses MitoSense Red (a red,
laser-excitable dye) to monitor mitochondrial membrane
potential changes in early apoptosis, and 7-AAD (a live-cell-
impermeant DNA intercalator) to simultaneously monitor
cell membrane permeability changes in late apoptosis and
necrotic cell death.
FlowCellect MitoLive KitIncludes:
• MitoSense Red, a fluorescent cationic dye that
accumulates in the mitochondria and is responsive to
mitochondrial potential changes
• Calcein acetoxymethylester (calcein-AM) a non-
fluorescent, cell-permeant compound that is hydrolyzed
by intracellular esterases into the fluorescent anion
calcein and provides a measure of cellular vitality.
The simultaneous use of the reagents enables researchers
to obtain information on early and late apoptosis in one
simple assay.
FlowCellect MitoStress KitIncludes:
• MitoSOX™ Red, a live-cell-permeant, fluorogenic dye which
targets the mitochondria and reacts with superoxide
radicals and fluoresces yellow/red
• Annexin V conjugated to CF647 which binds to
phosphatidylserine (PS) on the surface of apoptotic cells
The simultaneous use of these reagents enables
researchers to obtain information on oxidative stress and
apoptosis in one simple assay.
FlowCellect Cytochrome c KitIncludes a directly labeled anti-Cytocrome c–FITC antibody
and Anti-IgG1-FITC isotype control. Viable or live cells will
demonstrate higher levels of Cytochrome c staining while
apoptotic cells which have released their Cytochrome c
from the mitochondria to the cytoplasm will demonstrate
reduced staining intensity. The FlowCellect Cytochrome c
flow cytometry kit is a simple, gentle, and fast method to
assess levels of Cytochrome c in mitochondria, providing a
valuable tool for assessing proapoptotic signaling and the
efficacy of proapoptotic anti-cancer agents in cells.
ORDERING INFORMATION FlowCellect Mitochondrial Kits
Description Qty/PkCatalogue No.
FlowCellect MitoPotential Red Kit • Two dyes for measuring cell death
and mitochondrial membrane potential
• MitoSense Red (Red Laser) / 7-AAD (Blue Laser)
100 tests FCCH100105
FlowCellect MitoDamage Kit • Three dyes to assess
mitochondrial potential, stress, and cell death
• Mitosense Red (Red) / Annexin CF 488 (Blue) / 7-AAD (Blue)
100 tests FCCH100106
FlowCellect MitoLive Kit • Two dyes to measure
mitochondrial health and cell vitality
• Mitosense Red (Red) / Calcein-AM (Blue)
100 tests FCCH100107
FlowCellect MitoStress Kit • Understanding the regulation of
apoptosis and oxidative stress.• MitoSox Red (Red) / Annexin V 647
(Blue)
100 tests FCCH100109
FlowCellect Cytochrome c Kit• Easy way to detect loss of
mitochondrial Cytochrome c in cells
• Anti-Cytochrome c (Blue) / IgG1 (Blue)
100 tests FCCH100110
FlowCellect Oxidative Stress Characterization Kit• Detection of oxidative stress by
flow cytometry• Anti-DNP (Blue)
25 tests FCCH025111
guava Mitochondrial Depolarization Assay Kit• Monitoring changes in
mitochondrial membrane potential• JC-1 (Blue) / 7-AAD(Blue)
100 tests 4500-0250
APOPTOSISCells respond to specific apoptotic signals by initiating
intracellular processes that result in characteristic
physiological changes. Among these changes are
externalizations of phosphatidylserine to the cell surface,
depolarization of mitochondrial membranes, cleavage
and degradation of specific cellular proteins, compaction
and fragmentation of nuclear chromatin, loss of cell
membrane integrity, and cellular shrinkage. Suppression or
enhancement of apoptosis is known to cause or contribute
to diseases such as cancer and diabetes, making the
apoptotic pathway a popular drug target.
Because apoptosis is a dynamic event, and the time
period during which cells exhibit apoptosis markers is
variable and short, flow cytometry is an ideal technique
for tracking cells through apoptosis. Our easy-to-use kits
enable you to examine cells at each of the various stages
of apoptosis.
Early Apoptosis Flow Cytometry Kits Two separate dyes identify a broad spectrum of
apoptotic and non-apoptotic cells: Annexin V binds to
phosphatidylserine on the external membrane of apoptotic
cells, while 7-AAD permeates and stains DNA of late-stage
apoptotic and dead cells.
Mid-Apoptosis Flow Cytometry Kits Caspase activity is measured using a FLICA (fluorescent
labeled inhibitor of caspase) reagent, supplemented by
a nuclear DNA stain 7-AAD, which evaluates membrane
integrity and cell viability. The assays are available in two
forms, sulforhodamine (SR) and carboxyfluorescein (FAM),
giving greater flexibility in assay design as well as the
capacity to multiplex caspase assays.
Late Apoptosis Flow Cytometry Kits The guava TUNEL assay detects apoptosis-induced DNA
fragmentation through a quantitative fluorescence assay.
Terminal deoxynucleotidyl transferase (TdT) catalyzes the
incorporation of bromo-deoxyuridine (BrdU) residues into
the fragmented nuclear DNA at the 3’-hydroxyl ends. A
TRITC-conjugated anti-BrdU antibody then labels these
DNA fragments. The assay distinguishes two populations:
non-apoptotic cells (TUNEL-negative) and apoptotic cells
(TUNEL-positive).
Live/Healthy(non-committed)
cells
FLICA(-)PI(-) FLICA(+)PI(-) FLICA(+)PI(+) FLICA(-)PI(+)
Early/Mid-stage(committed) apoptotic
cells
Late stageapoptotic/dying
cells
Dead cells
C+C+C+
C+
C+C+ C+
C+PI+PI+
PI+PI+
PI+PI+
Live/Healthy(non-committed)
cells
FLICA(-)PI(-) FLICA(+)PI(-) FLICA(+)PI(+) FLICA(-)PI(+)
Early/Mid-stage(committed) apoptotic
cells
Late stageapoptotic/dying
cells
Dead cells
C+C+C+
C+
C+C+ C+
C+PI+PI+
PI+PI+
PI+PI+
Live/Healthy(non-committed)
cells
FLICA(-)PI(-) FLICA(+)PI(-) FLICA(+)PI(+) FLICA(-)PI(+)
Early/Mid-stage(committed) apoptotic
cells
Late stageapoptotic/dying
cells
Dead cells
C+C+C+
C+
C+C+ C+
C+PI+PI+
PI+PI+
PI+PI+
Live/Healthy(non-committed)
cells
FLICA(-)PI(-) FLICA(+)PI(-) FLICA(+)PI(+) FLICA(-)PI(+)
Early/Mid-stage(committed) apoptotic
cells
Late stageapoptotic/dying
cells
Dead cells
C+C+C+
C+
C+C+ C+
C+PI+PI+
PI+PI+
PI+PI+
Advantages• Two dye strategy : Detect various stages of apoptosis
within a one assay
• Mix-and-read assay: Get standardized results even with
multiple users
• All-in-one-kit : Spend less time before analysis
• Compatible with pairing with FITC or PE probes or other
probes in the green or yellow channels.
• Probe with other markers in green and yellow channels
with the FlowCellect Mitochondrial kits.
Early Mid LateGuava Nexin® Multicaspase Guava TUNELAnnexin Red Caspase 3/7, 8, 9 Assay
MitoPotential Red Dual CaspaseMitoDamageMitoStressMitoLiveCytochrome c
c
cc
c
c+
PS
PS
PS
PS
PS
PSc+
c+
c+
c+
c+
G...CG...C
C...GA...T
G...CC...G
A...T
A
GG...C
G...C
C...GA...T
G...CC...G
A...T
A
GG...C
G...C
C...GA...T
G...CC...G
A...T
A
G
TdT+
BrdUTP
DNA strand breaks due to
apoptosis
Add BrdUTPto 3’OH ends
Antibody labeledbreak sites
TRITC-Anti-BrdU
How the guava TUNEL Assay Works
Discover the power of flow cytometry for multiparametric apoptosis analysis.
Live/Healthy(non-committed)
cells
Early/Mid-stage(committed)
apoptotic cells
Late stageapoptotic/dying
cells
Dead cells
FLICA(-)PI(-) FLICA(+)PI(-) FLICA(+)PI(+) FLICA(-)PI(+)
DNA strandbreaks due to apoptosis
Add BrdUTPto 3’OH ends
Antibody labeledbreak sites
8
9
FlowCellect Annexin Red KitTwo Dyes to distinguish early Apoptosis from later stages
PS PSPS
PS
PS = phosphatidylserine
Healthy Cell Membranes
PSPSPSPS
Apoptotic Cell Membranes
PSPS
PSPS
PS
PS= 7AAD
Annexin CF647
PS PSPS
PS
PS = phosphatidylserine
Healthy Cell Membranes
PSPSPSPS
Apoptotic Cell Membranes
PSPS
PSPS
PS
PS= 7AAD
Annexin CF647
In the early stages of apoptosis, phosphatidylserine
molecules, which can bind to Annexin V, move from
the inner leaflet, to the outer leaflet of the plasma
membrane. A rapid, sensitive, and convenient assay to
monitor early and late apoptosis, the FlowCellect annexin
red kit includes recombinant Annexin V conjugated to
the red sensitive dye CF647, and 7-AAD (a live cell-
impermeant dye) to measure cell membrane integrity.
After staining cells with FlowCellect Annexin Red kit,
three populations of cells can be identified in this assay:
• Non-apoptotic cells: Annexin V(-) and 7-AAD(-)
• Early apoptotic cells: Annexin V(+) and 7-AAD(-)
• Late-apoptotic or dead cells: Annexin V (+) and
7-AAD(+)
Kit Description Laser Buffer Pack
Annexin V, CF 647 Red 10X Assay Buffer
7-AAD Blue
Fig. 7. Dot plots depicting Jurkat cells stained using the FlowCellect Annexin Red kit. Jurkat cells were untreated (Plot A), treated with 0.1 µM (Plot B) or with 3 µM staurosporine (Plot C), and then stained using the FlowCelllect Annexin Red kit. The percentage of apoptotic cells increased from 36% to 92% in response to a 30-fold increase in staurosporine concentration; however, only a small fraction (< 10%) of the cells showed evidence of cell death.
7-A
AD
B. C.
Annexin V
A.
Red
2 F
luor
esce
nce
(RD
2-H
Log)
2% 2%
85% 11%
Red2 Fluorescence (RED2-HLog)
100 101 102 103 104100
101
102
103
104
100
101
102
103
104
Red
2 F
luor
esce
nce
(RD
2-H
Log)
4%
60% 36%
Red2 Fluorescence (RED2-HLog)
100 101 102 103 104100
101
102
103
104
Red
2 F
luor
esce
nce
(RD
2-H
Log)
7%
5% 92%
Red2 Fluorescence (RED2-HLog)
100 101 102 103 104
3 µM Staurosporine0.1 µM StaurosporineUninduced
ORDERING INFORMATION Apoptosis Kits Description Qty/Pk Catalogue No.
Early Apoptosis Kits
FlowCellect Annexin Red Kit (Annexin V-CF647) 100 tests FCCH100108
guava Nexin Kit (Annexin V-PE) 100 tests 4500-0450
Mid-Apoptosis Kits (For a complete listing visit: millipore.com/midapoptosis_kits)
guava MultiCaspase SR Kit 100 tests 4500-0500
guava MultiCaspase FAM Kit 100 tests 4500-0530
guava MultiCaspase 3/7 FAM Kit 100 tests 4500-0540
guava MultiCaspase 3/7 SR Kit 100 tests 4500-0510
Late Apoptosis Kit
guava TUNEL Reagent Kit 100 tests 4500-0121
FEATURED PRODUCT
Signal transduction pathways lead to diverse outcomes,
such as apoptosis, cell differentiation, cell growth and cell
proliferation, all of which have been extensively studied in
the process of developing therapies for various cancers
and autoimmune disease. Cross-talk among signaling
pathways adds an extra dimension of complexity when
analyzing physiological consequences of a pathway of
interest. However, there are some key nodes at which
multiple signals are integrated. Multiparametric flow
cytometry provides researchers the power to monitor
these intracellular ‘checkpoints’ simultaneously, enabling
analysis of complicated cell events.
FlowCellect Cell Signaling Kits The study of cell signaling has been made easier by
activation status-specific and phospho-specific antibodies.
Measuring the activity of cell signaling pathways by flow
cytometry delivers robust, high content information in
less time than traditional methods by analyzing multiple
parameters on hundreds of cells per second.
Dual Detection FlowCellect Kits With Pairs of Total and Phospho-Specific Antibodies
We carefully selected pairs of antibodies that bind to
the same protein target; one to detect total protein
expression and another to detect the phosphorylated
from of the same protein. By using two-color
analysis we can achieve target-specific detection
of phosphorylation and, by doing so, eliminate false
positives in mixed cell populations. Performing dual
detection of treated cells reveals an accurate,
biologically relevant activation state for a given protein.
Implementing this technique can prove to be a powerful
tool in assessing small molecule activity and specificity
in drug screening campaigns.
AdvantagesAssay:• Ensures specific labeling of targets
• Multiplex detection with no optimization required
• Enables novice users to perform complex analysis
Samples:• Designed to run 25 samples of human cells
EGFR and MAP Kinase Kits• MAPK Activation Dual Detection Kit - Includes two
directly conjugated antibodies, anti-phospho-ERK1/2
(Thr202/Tyr204, Thr185/Tyr187)-PE and anti-ERK1/2-
Alexa Fluor 647 antibody to measure total levels of
ERK. This two-color flow cytometry kit is designed to
measure the extent of MAPK phosphorylation relative to
the total MAPK expression in any given cell population.
• PI3K/MAPK Dual Pathway Activation and Cancer Marker
Detection Kit
• EGFR/MAPK Pathway Activation Detection Kit
• EGFR RTK Activation Dual Detection Kit
PI3K / Akt / m-TOR Kits:• FlowCellect PI3K-mTOR Analysis Kit - This kit includes
two directly conjugated phospho-specific signaling
antibodies which are optimized for multicolor flow
cytometry to analyze the mTOR pathway in great
detail. The phosphorylation of Akt is indicative of the
upstream PI3K signaling, marking the cell’s initiation into
proliferation or cell survival. Phosporylated ribosomal S6
protein is indicative of downstream mTOR and p70S6K
signaling leading to protein translation.
• PI3K Activation Dual Detection Kit
Cell Signaling
Discover the power of flow cytometry for multiparametric cell signaling research.
FlowCellect Cell Signaling Kits With Directly Conjugated, Phospho-Specific Antibodies
Determine the effect of mechanical and chemical
reagents that can induce DNA damage, discern multiple
pathway activation and cross talk in a time-dependent
manner, or study the correlation between pathway
activation and changes in cell function and health.
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FlowCellect PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection KitThree antibodies to study cross talk between the PI3K and
MAPK pathways. The kit uses directly labeled antibodies
against phospho-Akt1/PKBα(Ser473) and phospho-Erk1/2
(Thr185/Tyr187) to analyze signaling activation and cross
talk, plus Ki-67 marker to identify the proliferative fraction.
Together, the antibody trio makes it easy to evaluate
the role these signaling pathways play in proliferation
and differentiation. The kit also includes Cell Cycle Stop™
fixation reagent to improve Ki-67 detection. Although Ki-67
is present throughout most of the cell cycle, it is difficult
to detect by flow cytometry except during M phase. Cell
Cycle Stop reagent arrests the cycle at the M phase,
making it possible to accurately detect Ki-67 expression
and discern the biological effects of the PI3K/MAPK cross
talk.
Kit Description Laser Buffer Pack
Anti-Phospho-Erk1/2 (Thr202/try204, Thr185/Tyr187)
Alexa Fluor 488
Cell Cycle Stop Fixation Buffer Wash Buffer Assay Buffer Permeabili-zation Buffer
Anti-Phospho-Ark1/PKBα (Ser473)
R-Phycoerythrin
Anti-Ki-67 PerCP
Fig. 8. HEK293 cells were stimulated by insulin (B, E) or PMA (C, F) for three or five minutes, and simultaneously stained with antibodies against phospho-Akt and phospho-ERK1/2. The cross-talk between the PI3K/MAPK signaling pathways is demonstrated by the sharp decrease in ERK phosphorylation after five minutes of insulin stimulation.
1040.97% 0.47%
0.30%
103
102
101
100
100 101 102 103 104
pAKT-Alexa 488
pER
K-P
E
98.26%
1040.78% 61.94%
30.37%
103
102
101
100
100 101 102 103 104
pAKT-Alexa 488
pER
K-P
E
6.91%
104 100%90%80%70%60%50%40%30%20%10%
0%pAkt pErk
Untreated100 nM Insulin 3 min100 µg/mL PMA 3 min
Ki67
30.30% 60.06%
1.28%
103
102
101
100
100 101 102 103 104
pAKT-Alexa 488
pER
K-P
E
8.35%
104 1.04% 0.31%
0.18%
103
102
101
100
100 101 102 103 104
pAKT-Alexa 488
A. Untreated B. Insulin Treated C. PMA Treated
D. Untreated E. Insulin Treated F. PMA Treated
HEK293
HEK293
3-M
inut
e Sti
mul
atio
n
3-Minute Stimulation
5-M
inut
e Sti
mul
atio
n
5-Minute Stimulation
pER
K-P
E
98.47%
104 0.72% 15.46%
75.15%
103
102
101
100
100 101 102 103 104
pAKT-Alexa 488
pER
K-P
E
8.67%
104 34.29% 49.52%
3.46%
103
102
101
100
100 101 102 103 104
pAKT-Alexa 488
pER
K-P
E
12.73%
100%90%80%70%60%50%40%30%20%10%
0%pAkt pErk Ki67
GTP
GDP
Shc
Grb2
Ki 67
PI3-Kinase
Proliferation/Survival
A CancerProliferative Marker
Growth/Differentiation
Wortmannin or LY294002
PMA
p85
IGF-1
p110 RAS
Raf-1Akt
P
P
P
P
P
SOS
MEK1/2
Inhibiting
Activating
ERK1/2
P
FEATURED PRODUCT
Fig. 9. Dual Parameter Analysis of Total and Phospho ERK 1/2 on Jurkat Cells. Analysis of total (x axis) and phospho (y axis) ERK on stimulated Jurkat cells stained with both anti-pERK1/2-PE and anti-ERK-Alexa Fluor 647 confirms target specificity of the phosphorylation event as indicated by the double positive cell population (9b). Stimulated Jurkat cells stained only with isotype control antibody showed no double positive cells (9a).
FlowCellect MAPK Activation Dual Detection Kit
Jak/STAT Kits• FlowCellect Multi-STAT Activation Kit – This kit is
designed to simultaneously detect the phosphorylation
of the most commonly studied STAT proteins through
cytokine or growth factor-mediated pathway activation.
The kit also enables a researcher to quickly profile
the status of constitutive activation of STAT1, STAT3,
and STAT5 within a population of cells or tumors. The
ability to detect the activation of multiple STAT proteins
simultaneously enables profiling of STAT signaling
pathways within a cell type of interest. Alterations in
these signaling profiles can be used to monitor the
effect of a particular upstream treatment targeting the
STAT pathways.
• EGFR/STAT3 Pathway Activation Detection Kit
DNA Damage Kit:Millipore’s FlowCellect multicolor DNA damage response
kit enables fast and easy detection of the phosphorylation
state of ATM, SMC1 and histone H2A.X by flow cytometry.
Millipore’s FlowCellect multicolor DNA damage response kit
was developed and tested using the DNA damaging reagent,
etoposide, in HeLa cells, but the kit can be used with other
human cell lines to determine the effect of mechanical and
chemical reagents that can induce DNA damage through the
ATM- dependent pathway.
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FEATURED PRODUCT
FlowCellect MAPK Activation Dual Detection KitMillipore’s FlowCellect MAPK activation dual detection
kit includes two directly conjugated antibodies, a
phospho-specific Anti-phospho-ERK1/2 (Thr202/Tyr204,
Thr185/Tyr187)-PE and an Anti-ERK1/2-Alexa Fluor 647
conjugated antibody to measure total levels of ERK. This
two-color flow cytometry kit is designed to measure the
extent of MAPK phosphorylation relative to the total
MAPK expression in any given cell. The levels of both
the total and phosphorylated protein can be measured
simultaneously in the same cell, resulting in a normalized
and accurate measurement of MAPK activation after
stimulation. Moreover, simultaneous measurement of both
total and phospho-ERK1/2 confirms target specificity of
the phosphorylation event. Together, a total and phospho
antibody duo used in multiplex provides an enhanced and
more reliable detection of the phosphor: total ratio within
a mixed population.
9a.
9b.
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ORDERING INFORMATION Chemokine Receptor Kits Description Qty/Pk Catalogue No.
Flowcellect Chemokine Receptor CCR2B Surface Expression Quantification Kit
100 tests
FCCR2004411
Flowcellect Chemokine Receptor CCR4 Surface Expression Quantification Kit
100 tests
FCCR4004413
Chemokine Receptor CCR6 Surface Expression Quantification Kit
100 tests
FCCR604414
Flowcellect Chemokine Receptor CCR7 Surface Expression Quantification Kit
100 tests
FCCR7004415
AdvantagesAssay:• Includes pharmacologically characterized positive
and negative control cells
• Same accuracy as radioactive assays, without the
hazards.
• Ability to identify high, medium, and low expressing
cell cultures during the clonal selection process.
• High reproducible results obtained by using
Millipore’s well-characterized ChemiScreen™ GPCR
cell lines as assay controls
Samples:• Sufficient reagents to run 100 samples of human
cells.
FlowCellect Chemokine Receptor Surface Expression Quantification KitsMillipore offers 11 GPCR surface identification flow
cytometry kits. Flow cytometry provides high quality,
reproducible data in far less time than traditional methods
for chemokine receptor research and avoids the hazard and
expense of radioligand binding assays.
Our FlowCellect GPCR identification kits can be used to
identify and quantify GPCRs on the surface of any cells.
The kits detect GPCR expression using a GPCR-specific
antibody validated for flow cytometry. Also included are
positive and negative control cells with well-characterized
receptor expression levels for the purposes of quantitative
extrapolation.
For a complete listing of kits visit: millipore.com/flowcytometry
ORDERING INFORMATION Cell Signaling Kits Description Qty/Pk Catalogue No.
PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection Kit• Anti-phospho-Erk1/2 (Thr202/Tyr204,
Thr185/Thr187)-R-Phycoerythin• Anti-phospho-Akt1/PKBα
(Ser473) – Alexa Fluor 488• Anti-Ki-67-PerCP
25 tests FCCH025100
Multicolor DNA Damage Response Kit• Anti-phospho-SMC1
(S957)- Alexa Fluor 488• Anti-phospho-ATM
(S1981)- PE• Anti-phospho-HistoneH2A.X
(S139)- PerCP
25 tests FCCH025104
EGFR/MAPK Pathway Activation Detection Kit• Anti-phospho-EGFR
(Tyr1173) – Alexa Fluor 488• Anti-phospho-Erk1/2 (Thr202/Tyr204,
Thr185/Thr187)-R-Phycoerythin
25 tests FCCS025101
PI3K Activation Dual Detection Kit• Anti-phospho-Akt
(Ser473)- Alexa Fluor 488• Anti-Akt/PKB-Alexa Fluor 647
25 tests FCCS025105
MAPK Activation Dual Detection Kit• Anti-phospho-Erk1/2
(Thr202/Tyr204, Thr185/Thr187)- PE• Anti-Erk1/2 –Alexa Fluor 647
25 tests FCCS025106
FlowCellect Bcl-2 Activation Dual Detection Kit
25 tests FCCSO25108
EGFR/STAT3 Pathway Activation Detection Kit • Anti-phospho-EGFR (Tyr1173)• Anti-phospho-pSTAT3 (Try705)
25 tests FCCS025111
FlowCellect p38 Stress Pathway Activation Detection Kit
25 tests FCCSO25132
PI3K-mTOR Signaling Cascade Mapping Kit• Anti-phospho-Ribosomal Protein S6
(Ser235) – PerCP• Anti-phospho-Akt1/PKBα
(Ser473) – Alexa Fluor 488
25 tests FCCS025210
Multi-STAT Activation Profiling Kit• Anti-phospho-STAT1
(Y701)- PerCP• Anti-phospho-STAT3
(Y705)- Alexa 488• Anti-phospho-STAT5A/5B
(Y694/Y699)- PE
25 tests FCCS025550
EGFR RTK Activation Dual Detection Kit• Anti-phospho-EGFR
(Tyr1173)- Alexa Fluor 488• Anti-EGFR-PerCP
25 tests FCCS025107
Because flow cytometry has the power to characterize
subpopulations of cells within heterogenous cell mixtures;
it is widely used for studying both embryonic stem cells
(ESCs) and induced pluripotent stem (iPS) cells. Flow
cytometry enables researchers to evaluate percentages
of cells expressing specific markers, to determine culture
quality, and to track gene expression changes during a
differentiation protocol.
Embryonic Stem Cells (ESC)
Stem cell
Committed cell
Differentiated cells
(hESC, mESC)
(ENStem-A)Oct-4+
SSEA-4+ (human)TRA1-60+
HESCA+
Nanog+
SSEA-1+ (mouse)
Oct-4-/+
SSEA-4-
TRA1-60-
HESCA-
Oct-4-
SSEA-4-
TRA1-60-
HESCA-
SSEA-1+/-
FlowCellect Stem Cell Characterization KitsMillipore’s FlowCellect stem cell characterization kits
are designed to provide rapid, sensitive assessments of
embryonic and neural stem cell phenotypes at various
stages. The kits use three parameters for accurate
identification, enabling the research to “triangulate” cellular
phenotypes with two complementary positive markers and
one negative marker. The negative antibody also serves as
a stage- and species-specific or lineage-specific marker for
differentiated cells.
ORDERING INFORMATION Stem Cell Kits Description Qty/Pk Catalogue No.
Mouse Embryonic Stem Cell Nuclear Marker Characterization Kit• OCT-4, SSEA-1, SSEA-4
25 tests
FCMEC25110
Human Embryonic Stem Cell Nuclear Marker Characterization Kit• OCT-4, SSEA-4, SSEA-1
25 tests
FCHEC25102
Human Embryonic Stem Cell Surface Marker Characterization Kit• Tra-1-60, SSEA-4, SSEA-1
25 tests
FCHEC25106
Human Embryonic Stem Cell Surface Marker Characterization Kit• HESCA-1, SSEA-4, SSEA-1
25 tests
FCHEC25104
Rodent Neural Stem Cell Characterization Kit(Neural Differentiation)• SOX-2, Nestin, β-III-Tubulin
25 tests
FCRNC25112
Rodent Neural Stem Cell Characterization Kit(Astrocyte Differentiation)• SOX-2, Nestin, GFAP
25 tests
FCRNC25114
AdvantagesAssay:• All optimized, fluorescently-labeled flow cytometry
antibodies and buffers included
• Highly reproducible results
• Validated for flow cytometry, immunocytochemistry,
and Western blotting
Stem CellsDiscover the power of flow cytometry for multiparametric stem cell analysis.
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FlowCellect Human Embryonic Stem Cell Characterization Kit The kit is provides a quick, easy way to track surface marker expression of hOCT4, SSEA4 and SSEA1. The
percentage of undifferentiated human embryonic stem cells in culture is reflected in the percentage cells
that express both hOCT4 and SSEA4 but not SSEA1. This quick test will enable researchers to determine the
multipotency of their cells in culture as well as see changes in marker expression during a differentiation protocol.
FlowCellect Human Embryonic Stem Cell HESCA-1 Surface Marker Characterization KitThis kit provides an easier and quick way to track surface marker expression of HESCA-1, SSEA4 and SSEA1.
The kit will also help to determine the percentage of undifferentiated human embryonic stem cells in culture by
determining the percentage of cells that express both HESCA-1 and SSEA4 and not SSEA1. This quick test will
enable researchers to test the quality of their cells in culture as well as see changes in marker expression during a
differentiation protocol.
Fig. 10. Representative data of H1 human embryonic stem cells stained with hOCT4-Alexa 488, SSFA4-PE and SSFA1-PF/CY5
10e40%
4.9%
0.1%
95%
10e3
10e2
10e1
10e010e0 10e1 10e2 10e3 10e4
SSEA
1-PE
/CY5
SSEA4-PE
A.
10e485.5%
14.4%
0.1%
0%
10e3
10e2
10e1
10e010e0 10e1 10e2 10e3 10e4
hOCT
4-Al
exa
488
SSEA1-PE/CY5
B. 10e40.1%
4.8%
85.5%
9.6%
10e3
10e2
10e1
10e010e0 10e1 10e2 10e3 10e4
hOCT
4-Al
exa
488
SSEA4-PE
C.A.
Fig. 11. Representative data of H1 human embryonic stem cells stained with HESCA-1-FITC, SSEA4-PE and SSEA1-PE/CY5.
B. C.A.
FEATURED PRODUCTS
The immune system, which mediates the body’s response
to the introduction of foreign material, is made up
of multiple cell types collectively called lymphocytes.
Lymphocyte subtypes include B cells (which secrete
antibodies), cytotoxic T cells, helper T cells (which secrete
cytokines), and natural killer (NK) cells. Characterization of
lymphocyte subtypes and cytokine signaling is essential for
understanding the complex nature of the immune system.
Activation by antigens, suppression of normal immune
activation, and disease states can affect the phenotypes
of lymphocytes. Multiparameteric phenotypic analysis
by flow cytometry allows researchers to distinguish one
subpopulation of cells within a heterogeneous mixture,
and thereby enables the study the dynamics of immune
signaling in intact cells.
FlowCellect Immunology KitsEach kit includes multiple optimized fluorescent labeled
antibodies and all buffers necessary for cell preparation
and analysis. Detailed assay instructions are included
to assist in analysis and to ensure that the correct cell
concentration is obtained during acquisition of sample data.
Fig. 12. Schematic diagram of CD4+ T-cell differentiation. Five different types of CD4+ T-cells can develop from a common naïve precursor depending on the cytokine environment and interaction with dendritic cells (DC).
AdvantagesAssay:• Multiplex detection with no optimization required
• Highly reproducible
• Minimal assay development needed
• All optimized flow cytometry antibodies and buffers
included
• Enables novice users to perform complex analysis
Samples:• Designed to run 25 tests (FlowCellect kits)
• Designed to run 100 tests (guava kit)
DC
IL-12RTH1T-betSTAT4STAT1
IL-2RTREG
Foxp3STAT5
IL-21RTFHTHBcl-6
STAT3
IL-4RTH2GATA3STAT6
STAT5a
IL-23RTH17
RORγtSTAT3
IL-12
CD80
CD28
TCR
CD28
CD85
pMHCII
IFNγIL-18
IL-2IL-4IL-33
IL-6
IFNγ
IL-2
LTα
IL-21
IL-6TGFβ
IL-23
TGFβIL-2
IL-4
IL-5
IL-13
IL-25
IL-17
IL-17F
IL-22
IL-21
IL-21
IL-17
TGFβ
IL-10
IL-35
B ZONE
CD4+ T-Cell Differentiation
CD62Llo CCR7lo
CCR7lo
High TCR Binding
Low TCR Binding
Medium TCR Binding
CXCR5hi
CD62Lhi CCR7hi T ZONE
EMIGRANTnaive
ImmunologyDiscover the power of flow cytometry for multiparametric analysis of the immune system.
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FlowCellect Human Lymphocyte ZAP-70 Characterization KitIncludes three directly conjugated antibodies: CD5 FITC,
CD19 APC, and ZAP-70 PE to provide researchers the ability
to phenotypically distinguish cell types. CD5 is normally a
pan-T-cell marker, while CD19 is a mature B-cell marker.
Some B-cell malignancies express both of these molecules
on their surface, and therefore can be distinguished from
the two aforementioned cell types. ZAP-70 is a molecule
involved in T-cell activation, which can also be expressed
within B-cells at levels correlating to disease severity.
The combinatorial staining of cells for all three of these
markers assists the researcher studying the molecular
biology of T-cell activation or B-cell disease states.
Fig. 13. Mixture of Roamos (B cell line) and Jurkat (T cell Line) cells were used as a model system. The cells were then stained with CD5-FITC, CD19-APC, and ZAP-70PE. By drawing two gates on the dot plot showing CD5 vs. CD19, the expression levels of each of the three mark-ers were determined for the two cell subpopulations.
FlowCellect Mouse FoxP3 Treg Identification KitThis kit provides a quick and easy way to detect FoxP3
expression in mouse CD4+ T-cells. T regulatory (Treg) cells
represent 2 - 10% of the total peripheral CD4+ T-cells and
are characterized by the expression of the forkhead box
transcription factor, FoxP3. FoxP3 is a master regulator of
Treg development and function. Treg cells develop from
a common naïve CD4+ T-cell precursor and expression of
FoxP3 is induced in the presence of TGFβ and IL-2. Lack of
FoxP3 in humans leads to the fatal autoimmune disease,
IPEX. In addition, studies with the FoxP3-deficient mouse
model, also known as the scurfy mouse, have shown that
FoxP3 expression is profoundly important for the regulation
of the immune system and the development of Treg cells.
guava Cell Toxicity Kit Obtain all relevant statistics, including percentages of
target cells killed, effector and target cell percentages,
and viability data with this convenient kit. The assay uses
a well-characterized cell tracking dye that is optimized for
use on all guava easyCyte instruments. The dye diffuses
freely into cells and is retained within the cell without
affecting cellular function.
ORDERING INFORMATION Immunology Kits Description Qty/Pk Catalogue No.
FlowCellect Human FoxP3 Treg Characterization Kit
25 tests
FCIM025118
Flowcellect Human Lymphocyte ZAP-70 Characterization Kit • Anti-CD5 / Anti-CD19 / Anti-
ZAP-70
25 tests
FCIM025122
FlowCellect Mouse FoxP3 Treg Identification Kit• Anti-CD4 / Anti-FoxP3 clone 3G3
25 tests
FCIM025126
guava Cell Toxicity Reagents Kit• guava CFSE / 7-AAD
100 tests
4500-0230
FEATURED PRODUCTS
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Advance your flow cytometry analysis with Millipore’s
growing selection of directly conjugated primary antibodies.
As a part of our complete benchtop flow cytometry solution
including instruments, software, service, and reagents,
Milli-Mark fluorescently-labeled antibodies are specifically
designed, optimized and validated for flow cytometry
applications.
Fig. 14. Fixed and permeabilized 2102Ep embryonal carcinoma cells were stained with Milli-Mark anti-hOct4-Alexa Fluor 488 (FCMAB113A4, green histogram) or IgG-Alexa Fluor 488 (grey histogram) at 1:100 for 1 hour and then analyzed by flow cytometry.
Fig. 15. Staurosporine-treated (yellow histogram) or untreated (grey histogram) Jurkat cells were stained using Milli-Mark pBcl2-(S70)-PE antibody (FCMAB140P) and analyzed using flow cytometry.
ORDERING INFORMATION Milli-Mark Stem Cell
Description Reactivity HostQty/Pk
Catalogue No.
Anti-hOCT4-Alexa Fluor 488
HumanMouse
Mouse 100 tests
FCMAB113A4
Anti-SSEA-4-PE Human Mouse 100 tests
FCMAB116P
Anti-Sox2-FITC HumanMouseRat
Mouse 100 tests
FCMAB112F
Anti-TRA160-FITC Human Mouse 100 tests
FCMAB115F
Anti-HESCA-1-FITC HumanMouse
Mouse 100 tests
FCMAB111F
Anti-SSEA-1-PE HumanMouseRat
Mouse 100 tests
FCMAB117P
Anti-SSEA3 Alexa 488, clone MC-631
Human Rat 100 tests
FCMAB141A4
Anti-Human Nuclei -FITC, clone 3E1.3
Human Mouse 100 tests
FCMAB157F
Anti-BMP-7-FITC, clone 2A10
Human Mouse 100 tests
FCMAB135F
Anti-mOCT4-Alexa Fluor 488, clone 7F9.2
HumanMouse
Mouse 100 tests
FCMAB124A4
Anti-TRA-1-81-FITC,clone TRA-1-81
Human Mouse 100 tests
FCMAB132F
Anti-TRA-2-49-FITC,clone TRA-2-49/6E
Human Mouse 100 tests
FCMAB133F
Anti-SRF-FITC, clone 1E1
Human Mouse 100 tests
FCMAB137F
ORDERING INFORMATION Milli-Mark Apoptosis and Cancer
Description Reactivity HostQty/Pk
Catalogue No.
Anti-phospho-Bcl-2 (Ser70)-PE, clone 69-10C-2-10C-18
Human Rabbit 100 tests
FCMAB140P
Rat-α-Caspase-2-FITC Human Rat 100 tests
FCMAB158F
For a complete listing of Milli-Mark Antibodies visit: millipore.com/antibodies and search keyword, “Milli-Mark.”
Apoptosis & Cancer
Milli-Mark Conjugated Antibodies
18
Fig. 17. HeLa cells were stained with either Milli-Mark anti-Syntaxin-3-FITC (FCMAB155F, green histogram) or IgG1 isotype control (grey histogram) and analyzed by flow cytometry.
Fig. 16. HeLa cells were stained with either Milli-Mark anti-Y14-FITC, clone 4C4 (FCMAB151F, green histogram) or with IgG2b isotype control antibody (grey histogram) and analyzed using flow cytometry.
ORDERING INFORMATION Milli-Mark Epigenetics and Gene Regulation
Description Reactivity HostQty/Pk
Catalogue No.
Anti-MAD2A-FITC, clone 17D10
Human Mouse 100 tests
FCMAB150F
Anti-Y14-FITC, clone 4C4
Human Mouse 100 tests
FCMAB151F
Anti-FXR1-FITC, clone 6BG10
Human Mouse 100 tests
FCMAB152F
Anti-SMN-FITC, clone 2B1
Human Mouse 100 tests
FCMAB153F
Anti-CAF1 p150-FITC, clone SS1, 1-3
Human Mouse 100 tests
FCMAB145F
Anti-TBX21/ T-Bet-FITC
Human Mouse 100 tests
FCABS131F
Anti-RPA2 p34 -FITC, clone RPA20 1-46
Human Mouse 100 tests
FCMAB143F
Anti-RPA1 p70 -FITC, clone RPA9, 1-30
Human Mouse 100 tests
FCMAB144F
Anti-BMI1-FITC, clone AF27
Human Mouse 100 tests
FCMAB149F
Anti-RBMS1-FITC,clone 4D11
Human Mouse 100 tests
FCMAB123F
Anti-c-Jun-FITC, clone 6E4
HumanMouseRat
Mouse 100 tests
FCMAB122F
Anti-RBMS1-FITC,clone 4D11
HumanMouse
Mouse 100 tests
FCMAB125F
Anti-BrdU-Alexa 488 Human Mouse 100 tests
FCMAB101A4
Phospho-ATM (Ser1981) - PE
HumanMouseRat
Mouse 100 tests
FCMAB110P
Phospho H3 (Ser10)- Alexa 488
Human Mouse 100 tests
FCMAB104A4
Phospho-SMC1 (Ser957) - Alexa 488
HumanBovineXenopus
Mouse 100 tests
FCMAB108A4
Anti-Cyclin B1 - PE HumanMouse
Mouse 100 tests
FCMAB102P
For a complete listing of Milli-Mark Antibodies visit: millipore.com/antibodies and search keyword, “Milli-Mark.”
Epigenetics & Gene Regulation
ORDERING INFORMATION Milli-Mark Neuroscience
Description Reactivity HostQty/Pk
Catalogue No.
Anti-Syntaxin-3 FITC, clone 1-146
Human Mouse 100 tests
FCMAB155F
Anti-Rapsyn-FITC, clone 1234
Mouse Mouse 100 tests
FCMAB134F
Neuroscience
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Fig. 18. Flow cytometry analysis of human lymphocytes stained with Milli-Mark anti-CD4-FITC (FCMAB170F).
Fig. 19. A431 cells were stained with either Milli-Mark anti-EGFR-PerCP (FCMAB129CP, red histogram) or isotype control (grey histogram) and analyzed by flow cytometry.
ORDERING INFORMATION Milli-Mark Inflammation/Immunology
Description Reactivity HostQty/Pk
Catalogue No.
Anti-CD1a-FITC, clone NA1/34
Human Mouse 100 tests
FCMAB166F
Anti-CD2-FITC, clone MT910
Human Mouse 100 tests
FCMAB167F
Anti-CD3-PECy5, clone UCHT1
Human Mouse 100 tests
FCMAB169C5
Anti-CD4-FITC, clone MT310
Human Mouse 100 tests
FCMAB170F
Anti-CD8-FITC, clone DK25
Human Mouse 100 tests
FCMAB176F
Anti-CD11c-FITC, clone KB90
Human Mouse 100 tests
FCMAB179F
Anti-CD13-FITC, clone WM-47
Human Mouse 100 tests
FCMAB180F
Anti-CD14-FITC, clone TUK4
Human Mouse 100 tests
FCMAB181F
Anti-CD16-FITC, clone DJ130c
Human Mouse 100 tests
FCMAB183F
Anti-CD19-APC, clone HD37
Human Mouse 100 tests
FCMAB185AP
Anti-CD27-FITC, clone M-T271
Human Mouse 100 tests
FCMAB191F
Anti-CD45-PE, clone F10-89.4
Human Mouse 100 tests
FCMAB118P
Anti-CD45RA-FITC, clone MEM 56
HumanMouse
Mouse 100 tests
FCMAB126F
Anti-CD56-PE, clone MOC-1
Human Mouse Mouse
100 tests
FCMAB200P
Anti-CD57-FITC, clone TB01
Human Mouse Mouse
100 tests
FCMAB201F
ORDERING INFORMATION Milli-Mark Cell Signaling
Description Reactivity HostQty/Pk
Catalogue No.
Anti-EGFR-PerCP, clone LA22
Human Mouse 100 tests
FCMAB129CP
Anti-mTOR-FITC, clone 2ID8.2
Human Mouse 100 tests
FCMAB154F
Anti-Ras-FITC, clone RAS10
Human Mouse 100 tests
FCMAB148F
Anti-GβL/mLST8, clone 3E1.2, FITC Conjugate
HumanMouse
Mouse 100 tests
FCMAB121F
Anti-Akt/PKB-Alexa Fluor 647, clone SKB1
Human Mouse 100 tests
FCMAB128A6
Phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187)-PE
HumanMouseRat
Rabbit 100 tests
FCMAB100P
Anti-Ki-67-APC Human Mouse 100 tests
FCMAB103AP
Phospho-STAT1 (Y701) Alexa 488
HumanMouseRat
Mouse 100 tests
FCMAB106A4
Anti-β-catenin-PE, clone 7F7.2
Mouse Mouse 100 tests
FCMAB1209P
Phospho STAT5A/B (Y694/699) –PEH
HumanMouseBovineSheep
Mouse 100 tests
FCMAB105P
Inflammation/Immunology Cell Signaling
For a complete listing of Milli-Mark Antibodies visit: millipore.com/antibodies and search keyword, “Milli-Mark.”20
InstrumentsThe guava easyCyte flow cytometry systems are
uncomplicated instruments that deliver complex cell
analysis—right on your benchtop. The culmination of over
a decade of flow cytometry expertise, these instruments
use smaller sample, generate less waste, and are easier to
use and maintain than traditional flow cytometers—all while
providing the power you need in the most compact format
available. These advantages are made possible by our
patented microcapillary flow cell technology.
Microcapillary Flow Cytometry TechnologyAt the heart of every guava easyCyte system is a unique,
microcapillary flow cell that eliminates the need for sheath
fluid. This translates into smaller samples, less reagents
and minimal waste—saving you both time and money.
Because the flow cell is self-aligning and user-replaceable,
you can remove it yourself at any time for cleaning and
maintenance—no more expense or downtime for service
visits. And by eliminating complicated fluidics, we’ve created
a tiny instrument footprint that fits into the tightest spots,
saving valuable laboratory space.
Components Of The
Guava Flow Cytometry
Solution
Sheath Fluid: ~10 mL/testWaste: 8,000 mL per 8 hour runTypical #Cells Per Protocol: 100,000 - 1,000,000 cells/test
Sheath Fluid: NoneWaste: < 80 mL per 8 hour runTypical #Cells Per Protocol: 1,000 - 10,000 cells/test
Traditional Microcapillary
Waste
SampleSheath Fluid
Laser
Traditional sheath fluid system Guava-patented microcapillary system
Laser
She
ath
Flow
Inje
ctio
n Tip
Waste
Sam
ple
Flow
Sample
Laser
WasteLaser
Waste
Flow Cell
Sample
Sam
ple
Flow
21
Small footprint saves valuable laboratory space
Width: 20.3 in (51.5 cm)
Depth: 23.4 in (59 cm)
Height: 10.0 in (25.4 cm) (does not include laptop)
Wash vial offers a high-pressure purge to easily clear obstructions from the flow cell
Waste vial collects less than 80 mL of waste in a typical 8-hour workday
Robotic sample tray provides walk-away automation for a 96-well microplate and up to 10 sample tubes
Microcapillary flow cell requires no sheath fluid and
is user-replaceable
Up to six-color detection made possible by one (blue) or
two excitation lasers (blue & red)
SPECIFICATIONS
System easyCyte 5HT easyCyte 6HT easyCyte 6HT-2L easyCyte 8HT
Catalogue # 0500-4005 0500-4005 0500-4007 0500-4008
Option # N/A 0500-4006 N/A N/A
Laser Blue (488 nm) Blue (488 nm)Blue (488 nm) and
Red (640 nm)Blue (488 nm) and
Red (640 nm)
FSC
SSC
Green 525/30 nm 525/30 nm 525/30 nm 525/30 nm
Yellow 583/26 nm 583/26 nm 583/26 nm 583/26 nm
Red1 680/30 nm 680/30 nm 690/50 nm 690/50 nm
NIR1 N/A 785/70 nm N/A 785/70 nm
Red2 N/A N/A 661/19 nm 661/19 nm
NIR2 N/A N/A N/A 785/70 nm
Microcapillary Fluidics
Direct, Absolute Cell Counts
Automation – 96 Well and 10 Tubes
Mixing
Dell® Latitude® E6500 Laptop with Intel® Core 2 Duo (P8600), 2.40 GHz, 1066 MHz FSB
InCyte™ Software
guavaSuite Software Modules
Digital Signal Processing
guava easyCyte High Thoughput Sampling Instruments
22
Small footprint saves valuable laboratory space
Width: 17.75 in (45.1 cm)
Depth: 17.25 in (44.5 cm)
Height: 8.75 in (22.2 cm) (does not include laptop)
Wash vial offers a high-pressure purge to easily clear obstructions from the flow cell
Single sample loader Swivel arm functionality, holds two tubes and allows instant acquisition
Waste vial collects less than 80 mL of waste in a typical 8-hour workday
Up to six-color detection made possible by one (blue) or
two excitation lasers (blue and red)
Microcapillary flow cell requires no sheath fluid and is
user-replaceable
SPECIFICATIONS
System easyCyte 5 easyCyte 6 easyCyte 6-2L easyCyte 8
Catalogue # 0500-5005 0500-5005 0500-5007 0500-5008
Option # N/A 0500-5006 N/A N/A
Laser Blue (488 nm) Blue (488 nm)Blue (488 nm) and Red
(640 nm)Blue (488 nm) and Red
(640 nm)
FSC
SSC
Green 525/30 nm 525/30 nm 525/30 nm 525/30 nm
Yellow 583/26 nm 583/26 nm 583/26 nm 583/26 nm
Red1 680/30 nm 680/30 nm 690/50 nm 690/50 nm
NIR1 N/A 785/70 nm N/A 785/70 nm
Red2 N/A N/A 661/19 nm 661/19 nm
NIR2 N/A N/A N/A 785/70 nm
Microcapillary Fluidics
Direct, Absolute Cell Counts
Automation – 96 Well and 10 Tubes
N/A N/A N/A N/A
Mixing N/A N/A N/A N/A
Dell Latitude E6500 Laptop with Intel Core 2 Duo (P8600), 2.40 GHz, 1066 MHz FSB
InCyte Software
guavaSuite Software Modules
Digital Signal Processing
guava easyCyte Single Sample Instruments
23
24
ORDERING INFORMATION Flow Cytometry SystemsDescription Catalogue No.
Single Sampling Instruments
guava easyCyte 5 Base System 0500-5005
guava easyCyte 6-2L Base System 0500-5007
guava easyCyte 8 Base System 0500-5008
PCA Base System 0500-1090
High Throughput Sampling Instruments
guava easyCyte 5HT Base System 0500-4005
guava easyCyte 6HT-2L Base System 0500-4007
guava easyCyte 8HT Base System 0500-4008
PCA-96 Base System 0100-8710
SOFTWAREYour specific research needs are always changing, and
Millipore’s guava optimized software modules are flexible
enough to accommodate you at every level. Because all our
software modules use the same, intuitive user interface, it’s
easy to switch from one to another. The guavaSoft suite is
best for user-defined assays, the assay-specific software
modules can provide the most focused data output, and
the InCyte software package enables easy visualization
of data in a broad biological context. You can export to
spreadsheets or any third-party analysis format, and easily
attain 21 CFR part 11 compliance with all our software
packages.
Drag-and-drop gating from one plot to another
View up to 11 plots at once
Real-time plot adjustments
Organize acquired data sets and select individual wells for display
Quickly link to and review previously analyzed data
Easily cre-ate analysis templates
Combine groups of data and analysis templates to construct heat maps or EC
50 curves
Slider bars set cut-offs or threshold values for each experimental sector
Heat map shows values across an entire plate
InCyte: IntuitiveInCyte software is the first analysis package designed
specifically to give every user the power to draw
conclusions about the biological significance of data. It has
an intuitive, easy-to-use interface that makes it possible
to visualize and compare up to eight data sets at the same
time. The software is ideally suited for interrogation of high
content data sets derived from multiple functional studies.
InCyte software brings a new level of analytical power to
flow cytometry, enabling users to analyze entire plates of
data in less time than it usually takes to analyze a single
sample. In addition, data acquisition is fully incorporated
so that InCyte software can function as the primary data
acquisition and analysis package for the instrument. Most
importantly, comparative results are displayed at the
experiment level rather than on an individual well/sample
basis.
Start your benchtop cell analysis at: millipore.com/flowcytometry.
Protocols, products, webcast links, online demos and complete support are one click away.
Millipore, Advancing Life Science Together, guava, ViaCount are registered trademarks of Millipore Coporation.The M mark, Cell Cycle Stop, ChemiScreen, easyCyte, InCyte and Milli-Mark are trademarks of Millipore Corporation.Alexa Fluor is a registered trademark and MitoSOX is a trademark of Life Technologies, Inc.Dell and Latitude are registered trademarks of Dell, Inc. Intel is a registered trademark of Intel Corporation.Lit No. PB3945EN00, 09/10 LS SBU-10-03539Printed in U.S.A. and France© 2010 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.
www.millipore.com/flowcapabilities
TO PLACE AN ORDER OR RECEIVE TECHNICAL ASSISTANCE In the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)
In Europe, please call Customer Service:
France: 0825.045.645
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English UK: 0870.900.46.45
For other countries across Europe and the world,
please visit www.millipore.com/offices.
For Technical Service, please visit www.millipore.com/techservice.