ab131381 –
GSK3b Total and pSer9
Human Flow Cytometry
Assay Kit
Instructions for Use
For the measurement of GSK3b total and phospho-Ser9 levels in fixed cells. This product is for research use only and is not intended for diagnostic use.
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Table of Contents
1. Introduction 3
2. Assay Summary 5
3. Kit Contents 6
4. Storage and Handling 6
5. Additional Materials Required 7
6. Preparation of Reagents 8
7. Sample Preparation 8
8. Assay Procedure 10
9. Data Analysis 11
10. Assay Performance and Specificity 12
11. Frequently Asked Questions 16
12. Troubleshooting 18
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1. Introduction
Principle: ab131381 is a panel of antibodies that measure the
protein levels of GSK3B total and phosphorylation of GSK3B at
serine residue 9. The assay combines the power of single cell
analysis obtained with flow cytometry and the specificity of
antibody-based immunostaining to quantitate protein and
phosphorylation levels in cultured cells. Cells are harvested and
fixed/permeabilized in suspension, targets of interest are detected
indirectly with highly specific, well-characterized monoclonal
antibodies that are then labeled with fluorescent antibodies.
This flow cytometry panel generates quantitative data with specificity
similar to Western blotting, but with much greater quantitative
precision. This method rapidly fixes the cells in-situ, stabilizing the
in-vivo levels of proteins, and thus essentially eliminates changes
during sample handling, such as with the preparation of protein
extracts.
Background:
Glycogen synthase kinase 3 is a proline directed serine, threonine
kinase originally identified due to its ability to phosphorylate and
inactivate glycogen synthase and later found to be of key importance
in signaling pathways, cell fate determination, energy metabolism,
transcription regulation, neuronal development and body pattern
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formation. There are two isoforms of GSK-3 (alpha and beta) which
show a high degree of homology within their catalytic domains.
Activity of GSK3-B is regulated by phosphorylation of serine 9
(inactivating), phosphorylation of tyrosine 216 (activating) and by
protein complex formation which can activate (Axin, APC, B-catenin)
or inactivate (Frat 1 and 2) the enzyme. GSK3-B regulates by
phosphorylation the activity of numerous metabolic and signaling
proteins such as glycogen synthase, ATP citrate lyase, cyclic-AMP-
dependent protein kinase, acetyl CoA carboxylase, cyclin D1, insulin
receptor substrate-1, pyruvate dehydrogenase amongst others.
Furthermore, it phosphorylates structural cytoskeletal proteins
(MAPs and tau), making it a key component of neuronal structure
and plasticity. GSK3-B is also important for the regulation of
transcription factors that modulate cell survival such as activator
protein-1, cyclic AMP response element binding protein (CREB),
Myc and NFkB.
Altered GSK3B function has been implicated in neuronal
dysfunction. Polymorphisms in the GSK3B gene have been shown
to be associated with an increased risk for Alzheimer disease and
with modifications to the disease risk in Parkinson’s disease due to
its interaction with microtubule associated protein tau (MAPT)
haplotypes. GSK3B has been found to be involved in the
development of psychiatric disorders after the findings that lithium
and valproate, both known to control bipolar disorder, are inhibitors
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of GSK3B. Furthermore the levels of GSK3B have been found
significantly reduced in post-mortem frontal cortex samples from
patients with schizophrenia. GSK3B has also been implicated in
diabetes, cancer and cardiac hypertrophy [PMID: 20331603].
GSK3B has become a key target of pharmacological development in
Alzheimer’s, cancer diabetes, obesity, asthma, immune disorders,
hypertension, AIDS and Glaucoma as indicated by filed patents in
the last decade.
2. Assay Summary
Harvest cells as a single cell suspension.
Fix cells with 4% paraformaldehyde for 15 minutes, pellet and wash.
Permeabilize/Block the cells for 30 minutes at RT then pellet.
Add primary antibodies diluted in 1X incubation buffer, incubate for 1
hour and wash.
Add secondary antibodies diluted in 1X incubation buffer, incubate
for 1 hour, wash and read with flow cytometer.
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3. Kit Contents
Item Quantity
10X Phosphate Buffered Saline (PBS) 100 mL
100X Triton X-100 1.25 mL
Blocking Buffer 10 mL
50X Antibody Cocktail:
Mouse Anti-GSK3B total +
Rabbit Anti-GSK3B pS9 Antibody
220 µL
4. Storage and Handling
Upon receipt, spin down the contents of the antibody cocktail vial.
Store all components upright at 4°C. This kit is stable for at least
6 months from receipt.
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5. Additional Materials Required
• Flow cytometer
• Microfuge
• Aspirator
• 20% paraformaldehyde
• Goat anti-mouse detection antibodies labeled with
fluorochrome(s) suitable for available flow cytometer
(e.g. Abcam, GAM IgG – H&L DyLight® 488 Cat#ab96879 )
• Goat anti-rabbit detection antibodies labeled with
fluorochrome(s) suitable for available flow cytometer
(e.g. Abcam, GAR IgG – H&L DyLight® 649 Cat# ab96902)
• Nanopure water or equivalent
• Optional for high throughput manipulations: Filter 96-well
plates with pore sizes smaller than the cell line in use
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6. Preparation of Reagents
6.1 Prepare 1X PBS by diluting 100 mL of 10X PBS in 900 mL
Nanopure water or equivalent. Mix well andstore at room
temperature.
6.2 Immediately prior to use prepare
2X Blocking-Permeabilization Buffer (2X Triton X-100,
20% Blocking Buffer in PBS.) Discard any excess.
6.3 Immediately prior to use prepare 1X Incubation Buffer
(10% Blocking Solution in PBS). Any excess should be
stored at -20˚C.
7. Sample Preparation
7.1 Cell culture and treatment conditions are dictated by the
experiment at hand. As a general guideline, it is advisable
to analyze at least 10,000 events (cells) on the flow
cytometer per sample/data point. Therefore at least four
to ten times that many cells should be collected per data
point to ensure sufficient material at the end of the
staining.
7.2 For suspension cells, generate a single cell solution by
pipetting up and down.
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7.3 For adherent cells, fully dissociate cells (e.g. trypsin) into
single cell suspension. Passaging the cell line the day
before the experiment onto a fresh culture plate may help
improve single cell dissociation on the day of the
experiment.
7.4 Maintain cells resuspended in the culture treatment media,
at approximately 1x106 cells per mL.
7.5 Overlay 20% paraformaldehyde on the cell suspension so
that the final concentration is 4%, gently mix by inverting
the tube and incubate at room temperature for 15 minutes.
7.6 Pellet cells at 350 - 500 x g for 5 minutes (depending on
cell size) and decant supernatant.
Note: paraformaldehyde is toxic: handle with care and
dispose of according to local requirements
7.7 Dislodge the pellet by gently tapping the bottom of the
tube and resuspend the cells in 1X PBS (1x105 per
0.05 mL) and aliquot into assay vials.
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8. Assay Procedure
Note: Provided reagents are sufficient for 100 tests in a 100 µL
volume or 200 tests in a 50 µL volume. Always include negative
controls as follows (1) – primary/+secondary and (2) – primary/-
secondary. After every centrifugation step, aspirate the
supernatant leaving 50 µL volume inside the assay vial and
gently tap the bottom of the tube to dislodge the pellet before
continuing to the next step.
8.1 Overlay an equal volume of 2X Blocking-Permeabilization
buffer into each assay vial (e.g. 50 µL of cell suspension +
50 µL of 2X Blocking-Permeabilization buffer) and
incubate for 30 minutes at room temperature.
8.2 Pellet cells at 350 - 500 x g for 5 minutes (depending on
cell size) and aspirate supernatant.
8.3 Prepare 50 µL (or 100 µL if using 100 tests) per assay
tube of 2X Primary Antibody Cocktail Solution in
1X Incubation Buffer (1:25 dilution of 50X Antibody
Cocktail) in order to overlay the 50 µL cell suspension (or
100 µL if using 100 tests) for a final 1X Antibody Cocktail
solution. Incubate at room temperature for at least 1 hour.
8.4 Pellet cells at 350 - 500 x g for 5 minutes (depending on
cell size) and aspirate supernatant.
8.5 Wash twice by centrifugation with 1 mL of 1X Incubation
Buffer per assay tube.
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8.6 Prepare 100 µL per assay tube of a Goat Anti-Mouse and
Goat Anti-Rabbit fluorochrome labeled cocktail solution in
1X Incubation Buffer per assay tube.
8.7 Add 100 µL of detector solution per assay tube. Incubate
for 1 hour at room temperature in the dark.
8.8 Pellet cells at 350 – 500 x g for 5 minutes (depending on
cell size) and aspirate supernatant.
8.9 Wash twice by centrifugation with 1 mL of 1X Incubation
Buffer per assay tube.
8.10 Add 100 µL of 1X PBS to each assay tube before reading
on the flow cytometer.
9. Data Analysis
Specific methods depend on the available flow cytometer. It is
important to appropriately establish forward and side scatter gates to
exclude debris and cellular aggregates from analysis. Certain
treatments may generate subpopulations of cells that are apparent
from the forward/side scatter plots. Under these circumstances it is
recommended to adequately gate the subpopulation of interest
before capturing events. If the histogram does not generate a
perfect normal distribution, use the median measurement to prevent
artifacts from skewing the data.
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10. Assay Performance and Specificity
Specificity
Assay specificity was demonstrated by using Jurkat cells grown in
RPMI media with 10% FCS and treated for 60 minutes with 100 nM
Calyculin or DMSO (vehicle control). Figure 1 shows flow cytometry
results of this cell-culture model.
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Figure 1. Antibody specificity demonstrated by Flow cytometry. Non-
induced Jurkat cells (red) and calyculin induced (blue) were targeted with
the antibody cocktail against GSK3B total and GSK3B (pSer9).
Background fluorescence (pink) was determined with a no primary-antibody
control. Primary antibodies were labeled with 1:500 dilution of GAM IgG –
H&L DyLight® 488 Cat# ab96879 and 1:500 dilution of GAR IgG – H&L
DyLight® 649 Cat# ab96902 respectively. Signal intensity was measured in
FL-1 (GSK3B total) and FL-4 (GSK3B pSer9). After background subtraction,
the calyculin induced cell line shows a 6 fold increase in the levels of
phosphorylated GSK3B in comparison to a 1.2 decrease in the levels of
GSK3B protein.
Confidence in antibody specificity is critical to Flow data
interpretation. Therefore, the antibodies in this kit were also tested
for specificity by fluorescence immunocytochemistry and western
blot using the same aforementioned cell-culture model. The images
below show induction of GSK3B phosphorylation at residue 9 both
by ICC and WB using the antibodies included in this panel.
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Figure 2. Antibody specificity demonstrated by immunocytochemistry.
ICC was carried out on HeLa cells treated with calyculin (left) or vehicle
(right) with Rabbit anti-GSK3B pSer9 and Mouse anti-GSK3B using all buffer
reagents as supplied in this kit. Labeling was carried out with 1:500 dilution
of GAR IgG - H&L DyLight® 594 Cat# ab96897 and 1:500 dilution of GAM
IgG – H&L DyLight® 488 Cat# ab96879 respectively. The calyculin induced
cells (left) show a significant induction of GSK3B phosphorylation at residue
S9 in comparison to the non-induced control (right).
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Figure 3. Validation of antibodies by WB. Western blot was run on a
4-20% gradient acrylamide gel. Samples were loaded as follows from left to
right: (1)50 ng GSK3B protein (ab63193), (2) 40 µg of Jurkat cells treated
with DMSO, (3) 40 µg of Jurkat cells treated with 100nM calyculin, (4) 40 µg
of HeLa cells treated with DMSO, (5) 40 µg of HeLa cells treated with
100 nM calyculin. Membrane blocking and incubation of primary and
secondary antibodies was carried out with 1X Blocking Buffer (ab126587) for
anti-GSK3B and anti-GSK3B pSer9. Actin was targeted with ab8224
following recommendations on product sheet.
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11. Frequently Asked Questions
11.1 The cells I am working with are extremely fragile and they
lyse during sample processing. How can I avoid this?
For extremely fragile cells we recommend to fix and
block/permeabilize the cells as suggested in this booklet
followed by a short staining procedure as follows:
11.1.1. Overlay 100 µL of 2X primary antibody cocktail
solution on cells suspension (in
Blocking/permeabilization buffer) and incubate for 1
hour at room temperature.
11.1.2. Centrifuge at 400g for 4 minutes and remove
supernatant. Do not wash.
11.1.3. Add 100 µL of 1x secondary antibody cocktail
solution and incubate for 1 hour at room
temperature in the dark.
11.1.4. Centrifuge at 400g for 4 minutes, remove
supernatant and wash once with 500 µL of
incubation buffer.
11.2 I grow my cells in 10% FBS, will this interfere with the cell
fixation?
Culture media containing up to 10% fetal serum does not
interfere with the cell fixation.
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11.3 How do I measure the assay background?
It is essential to omit primary antibody in at least one assay
vial to provide a background signal for the experiment which
can be subtracted from all measured data.
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12. Troubleshooting
Problem Cause Solution
Low Signal
Signal not correctly compensated
Check positive single color control is set up correctly
on flow cytometer and gated/compensated
correctly to capture all events
Insufficient secondary antibody
Increase concentration of antibody
Lasers not aligned Run flow check beads and
adjust alignment if necessary
Secondary antibody is not compatible
with primaries
Use secondary goat antibodies raised against mouse and rabbit for this
kit
High side scatter back-
ground
Cells lysed
Ideally samples should be freshly prepared. Do not
vortex or shake the sample at any stage. Do not exceed 500 x g for
centrifugation Bacterial
contamination Ensure sample is not
contaminated
Low event rate
Low number of cells Run 1x106 cells/mL
Cells clumped Ensure a single cell
suspension. Sieve clumps (30 µL nylon mesh)
High event rate
High number of cells/mL
Dilute between 1x105
cells/mL and 1x106
cells/mL
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