bd accuri™ c6 october 2015 - bnitm · october 2015. single cells in suspension pass a laser beam...
TRANSCRIPT
Flow cytometry workshopBD Accuri™ C6
October 2015
Single cells in suspension pass a laser beam
The cells produce characteristical light signals depending on cell type and preparation that will be analyzed by specific detectors
Priciple of flow cytometry
Algae
Bloodcells
DNA/RNA
Bacteria
What can be analyzed?
• Its relative size (Forward Scatter - FSC)
• Its relative granularity or internal complexity (Side Scatter - SSC)
• Its relative fluorescence intensity
Which parameters can be analyzed?
Forward Scatter—diffracted light Related to cell surface area Detected along axis of incident light in the forward direction
Side Scatter—reflected and refracted light Related to cell granularity and complexity Detected at 90° to the laser beam
Right Angle Light Detector
Cell Complexity
Forward Light Detector Cell Surface Area
Incident Light
Source
Properties of FSC and SSC
Basophil Granulozyte Lymphozyte
Eosinophil Granulozyte
Neutrophil Granulozyte
Monozyte
Thrombozytes
Erythrozytes
Granulocytes
Lymphozytes
(FSC)
Monozytes
Debris(S
SC)
Human whole blood
Lysed whole blood
The fluorochrome absorbs energy from the laser. The fluorochrome releases the absorbed energy by:
vibration and heat dissipation. emission of photons of a longer wavelength.
= 488 nm
Emitted Fluorescent Light Energy
Antibody
IncidentLight Energy
FluoresceinMolecule
520 nmHO
CO2H
O
C
What is fluorescent light?
Red laser 635 nmBlue laser 488 nm
FITC
488 nm 635 nm
PerCP
PE
APC
Excitation spectra
Blue laser
Red laser
Emission spectra
Relative fluorescence intensity
Num
ber o
f eve
nts
FITC
100 101 102 103 104FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Emitted fluorescence intensity proportional to binding sites
Fluorescence intensity
|480
|580
|560
|500
|520
|540
|620
|600Wave length(nm)
FITC
PE
530/30 585/42 488nm
100-
80-
60-
40-
20-
0-
Rel
ativ
e in
tens
ityFluorescence spillover
Increase of percentage
Compensation
The BD Accuri™ C6 Flow Cytometer
An affordable, full-featured, easy-to-use flow cytometer.Two lasers and six detectors
Components of a flow cytometer
• Fluidic system
• Optical system (excitation and detection)
• Electronic system and software
Fluidics
• Non-pressurized, peristaltic pump-driven system
• Patented pulse dampeners• User adjustable flow rate and
core diameter for a variety of applications
• Volume measurement for absolute counts
• Minimum sample volume 50 µL• Up to 10,000 events/second
Fluidics: Enhanced sample handling
• Microprocessor-controlled peristaltic pumps enable direct volume measurement
• Many types of sample tubes may be used• Falcon “FACS” tubes• Eppendorf• Micro-capillary tubes
• Add reagents or cells during a run• BD CSampler™ Accessory for automated
sample introduction
Fluidics: Sample Flow
Low pump speed High pump speed
Sample
LaminarFlow
Sheath SheathSample
High 66µl/min
LaminarFlow
Sheath Sheath
Low14µl/min
Excitation optics consist of: A laser Lenses to shape and focus the laser beam
Collection optics consist of: A collection lens to collect light emitted from the particle-
laser beam interaction A system of optical mirrors and filters to route specified
wavelengths of emitted light to designated optical detectors
Optics
460 500 540 460 500 540
LP 500 BP500/50
Longpass Bandpass
Optical filters
Signal amplification:• Photo diode• PMT (Photomultiplier Tube)
Optics: Detecors
Optics: System overview
FSC
SSC
488 nm solid state laser
640 nm diode laser
PMTs for fluorescence
Detection
Diodes for scatter detection
Compact optical system design reduces cost and
eliminates alignment issues
510/15540/20565/20610/20780/60
Instrumentconfiguration3 blue 1 red
User changeable
optical filters
BD Accuri™ C6 default optics
DetectorFL1
FL2
FL3
FL4
Filter533/30nm
585/40nm
670nm LP
675/25nm
FluorochromeFITC
PE
PerCP, PerCP-Cy5.5
APC
ColorGreen
Yellow/Orange
Dark Red
Red
“3 blue 1 red”
8-Peak Validation Beads (FL1, FL2, FL3)
6-Peak Validation
Beads (FL4)
FL1 = 533/30 FL2 = 585/40 FL3 = 670LP
101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106
FL4 = 675/25
101 102 103 104 105 106
Detectors – balanced at manufacture
39,27139,271Event 1Event 2Event 3
FSC SSC FITC PE0 60 120 90
10 160 65
30 650 160
FCS data
185Time
19002,688
31003,189
PE-A
1900
90
185 3100
PE-A
FITC
-A
1900
90
FITC
-A185 3100
Electronics: the result
Pre-optimized detectors – adjust view
Lymphocytes
Zoom Area
Zoom OutZoom Again
Optics: Advantages of pre-optimized voltage
• Greatly reduces risk of lost data due to improper set-up
• No specialist training or dedicated operator required
• Predictable, reproducible analysis relative to sample type and application
• Saves time and sample
• Attenuation filters (for too-bright signals) give controlled signal reduction
• Predictable fluorescence spillover
Compensation depends on
• Instrument configuration (filter and lasers)
• Used colors in panel
• Voltage (instrument settings)
predictable spillover for panels with same colors
Predictable spillover
FITC PE PerCP PerCPCy5.5 PE-Cy7 APC
FL1 (533 BP) ---- 3.20 0.00 0.00 0.50 0.00
FL2 (585 BP) 7.50 ---- 0.00 0.00 1.50 0.00
FL3 (670 BP) 1.00 19.50 ---- ---- ---- 0.80
FL4 (675 BP) 0.00 0.00 3.00 12.00 0.00 ----
Suggested Compensation Values for the BD Accuri C6
Compensation Controls – Beads
Advantages of compensation beads– Easy to use
– Do not require cells
29
FITC
PE
BD Accuri™ C6 Software
Sample Grid
CytometerStatus
Fluidics Controls
Run Criteria
Threshold and Compensation
Real Time Updates
Analysis andGating Tools
RegionsQuadrant Markers
HistogramsDot plotsDensity plots
Plot Statistics
Software: using Templates
BD Templates on the web
http://www.bdbiosciences.com/go/templates
Options in Flow CytometryApplications and staining targets
IntranuclearTranscription factors
Cell surfaceLineage markerActivation markerCytokine & chemokine receptors
IntracytoplasmaticCytokine, chemokines, various proteins
Apoptosis & cell viability
Proliferation
Quantification in supernatant, plasma, serum or cell lysates:Cytokines, Chemokines, immunoglobulins or signaling proteins
Supernatant, plasma, serum or cell lysates:ELISA, ELISPOT and Western Blot
Intracytoplasmatic or intranuclearCell Signaling Proteins
Microbiologywater analysis, bioprocessingMolecular biologytransfection
Human PBMC: T cell phenotyping
Lymphocyte Gate
Isotype-FITC CD45RA-FITC
CD
8-PE
-Cy7
CD3-APC
CD
4-PE
CD3-APC Isotype-FITC CD45RA-FITC
Gate:CD3+ CD8+
Gate:CD3+ CD4+
T cell phenotyping T regs: CD4-FITC FOXP3-APC
Sample B, C6
FOXP3-APC
CD
4-FI
TC
FOXP3-APC
CD
4-FI
TC
Sample A, C6
Methods: Blood samples were stained with CD4-FITC, fixed, permeabilized, and stained with FOXP3-APC. The background present in the CD4+FOXP3+ gate shown was 0% for the CD4+-FITC, unstained-APC control (data not shown).
2.4%
1.8%
Cell Cycle Analysis with Propidium Iodide
G1
S G2/M
Sub - G1
Camphthotecin induced murine thymus Annexin V
609.525 1.600.0001.000.000
0228.572
100.000
SSC-A
FSC-A
A01 Thymus campto unstainedGate: [No Gating]
P139,2% P2
37,0%
P139,2% P2
37,0%
101 107.2102 103 104 105 106
101
107.2
102
103
104
105
106
Annexin V APC - FL4-A
PI - FL2-A
A01 Thymus campto unstainedGate: P1 and P2
R10,1%
R266,2%
R30,1%
P30,2%
R10,1%
R266,2%
R30,1%
P30,2%
101 107.2102 103 104 105 106
101
107.2
102
103
104
105
106
Annexin V APC - FL4-A
PI - FL2-A
A02 Thymus Campto PIGate: P1 and P2
R10,1%
R239,7%
R312,9%
P320,9%
R10,1%
R239,7%
R312,9%
P320,9%
101 107.2102 103 104 105 106
101
107.2
102
103
104
105
106
Annexin V APC - FL4-A
PI - FL2-A
A03 Thymus Campto Annexin APCGate: P1 and P2
R162,8%
R219,6%
R30,3%
P30,0%
R162,8%
R219,6%
R30,3%
P30,0%
101 107.2102 103 104 105 106
101
107.2
102
103
104
105
106
Annexin V APC - FL4-A
PI - FL2-A
A04 Thymus Campto PI AnnexinGate: P1 and P2
R134,2%
R219,2%
R332,7%
P31,0%
R134,2%
R219,2%
R332,7%
P31,0%
Assessment of Cell ViabilityBD Horizon™ Fixable Viability Stain 520
• Amine-reactive dye useful for live/dead discrimination
• Compatible with fixation and permeabilization protocols
• Optimized all common laser lines:–Blue laser: - FVS520 (FITC channel) - FVS570 (PE channel)
–Red laser: - FVS660 (APC channel)
Examples of multicolor flow cytometric analysis of phosphorylated Stat3 expression by “viable” activated PBMCs.
Stat3 (pY705) AlexaFluor® 647
only living cells no live cell discrimination
BD Horizon™ Fixable Viability Stain 520
SSC
38
T cells only 0.4%
DC + T cells1 : 4 52.4%
In Vitro T cell proliferation: T cells + dendritic cells
39
CFSE
Gate = CD3+ Cells% of CD3+
T cellsProliferating
Courtesy of: Reddy P, Sung Y. Department of Pediatrics, University of Michigan, Ann Arbor, MI
FSC
-A
Flexibility in optical configuration - filters
Detector Filter options Fluorophore
FL1 530/30 BP (standard) FITC, GFP, YFP, CFSE, AF-488
515/15 GFP
FL2 585/40 BP (standard) PE, PI
540/20 BP YFP
FL3 670 LP (standard) PerCP, PerCP-Cy5.5, PE-Cy5, PE-Cy7, PI, 7-AAD
610/20 BP RFP, PI, PE-CF594
FL4 675/25 BP (standard) APC, PE-Cy5, AF-647
“3 blue 1 red”
Applications: GFP/YFP
Separation of GFP and YFP signals
Standard Filters GFP /YFP Combo
FL2:
585
/40
FL2:
540
/15
GFPYFP GFP
YFP
Detection of GFP expression in bacteria
Courtesy of: Tim F. Cooper, Dept. of Biology and Biological Chemistry, University of Houston, Houston,TX.
+ GFP
- GFP
E. coli B mixture
E. coli B+ GFP
plasmidE. coli B wild
type
FL1: 530 BP FL1: 530 BP FL1: 530 BP
Applications: Human Th1/Th2/Th17 CBA Kit
Optics: Selectable Lasers Module
FSC
SSC
510/15540/20565/20610/20780/60
3 blue 1 red2 blue 2 red
4 blue
User changeable optical filters
Selectable Lasers
Selectable Lasers Module provides flexibility
Reassign laser and detector associations
Standard: 488 FL1,2,3 640 FL42-blue 2-red: 488 FL1,2 640 FL3,44-blue: 488 FL1,2,3,4
FL1 FL2 FL3 FL4Standard: FITC PE PerCP-Cy5.5 APC2-blue 2-red: FITC PE APC-Cy7 APC4-blue: FITC PE PE-Cy5 PE-Cy7
Optical configurations
Detector Filter options Fluorophore
FL1 530/30 BP FITC, GFP, YFP, CFSE, AF-488
515/15 GFP
FL2 585/40 BP PE, PI
540/20 BP YFP
FL3 780/60 BP APC-eFluor-780, APC-Cy7
FL4 675/25 BP APC, PE-Cy5, AF-647
“2 blue 2 red”
Applications: BD™ CBA reagents
• BD CBA kits• Up to 7 analytes• Pre-defined panels
by area of biology• Th1/Th2• Th1/Th2/Th17• Inflammatory
cytokines• Chemokines• Anaphylatoxins
• BD CBA flex sets• Up to 30 analytes• User-defined panels
• Soluble proteins• Cytokines• Chemokines• Growth
Factors• Immunoglobulins• Enhanced
Sensitivity• <1.0pg/mL
Standard C6 configuration Selectable Lasers: 2-blue 2-red
Summary – The fluidics system
• Automatically starts up with the press of a single button.
• Self-cleans and alerts when fluids need attention.
• Is compatible with a variety of sample-tube configurations.
• Meters sample fluid uptake and cell counts
• Allows the user to independently adjust the speed of the sample uptake.
• Allows the user to adjust the core stream diameter for a range of cell sizes.
Summary – The optics system
• Is robust, making the instrument easy to transport.
• Allows the user to swap out interference filters easily.
• Requires no user alignment.
Summary – The electronics system
• Provides seven decades of dynamic range.
• Eliminates the need to adjust voltage settings.
• Provides software tools to help analyze a broad spectrum of data.
• Supports USB plug-and-play on a standard PC or laptop.
Summary – The software
• Can be used on any standard PC or laptop.
• Is intuitive to both novice and proficient users, even without training classes.
• Produces sophisticated graphics such as density plots and color histogram overlays.
• Uses the FCS 3.0 file format
• Allows users to drag and drop plot images into Microsoft Office applications
• and to copy and paste statistics to spreadsheet programs.
BD Accuri™ features that simplify multicolor analysis
• Digital system
• Four fluorescence detectors
• Flexibility in fluorochrome choice using:• Optional optical filters• Selectable Lasers option
• Predictable fluorescence spillover values
• Easy setup for data collection
• Validate system with beads
BD Accuri™ features that simplify multicolor analysis
www.bdbiosciences.com/support/resources/accuri
• Technical Bulletins
• BD Accuri C6 and BD Kits for Flow Cytometry
• BD Accuri C6 technical guidelines
• White papers
• BD Accuri proof sources
• User guides
• BD Accuri manuals
The BD Accuri™ C6 is Portable!
National Oceanic and Atmospheric Administration:Great Lakes Microcystis research project – Lake Erie
2nd Norwich Flow Day:Institute for Food Research
The Ecosystem Centre:Research vessel, Palmer Peninsula, Antarctica
Antarctica
Mobile Lab
Lake Erie