ab GSK3b Total and pser9 Human Flow Cytometry Assay Kit

Transcription

1 ab 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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3 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 Data Analysis Assay Performance and Specificity Frequently Asked Questions Troubleshooting 18 2

4 1. Introduction Principle: ab 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 3

5 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-ampdependent 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 4

6 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: ]. 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. 5

7 3. Kit Contents Item 10X Phosphate Buffered Saline (PBS) 100X Triton X-100 Blocking Buffer 50X Antibody Cocktail: Mouse Anti-GSK3B total + Rabbit Anti-GSK3B ps9 Antibody Quantity 100 ml 1.25 ml 10 ml 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. 6

8 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 7

9 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. 8

10 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 1x10 6 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 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 (1x10 5 per 0.05 ml) and aliquot into assay vials. 9

11 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 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 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. 10

12 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 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 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. 11

13 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. 12

14 Figure 1. Antibody specificity demonstrated by Flow cytometry. Noninduced 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. 13

15 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). 14

16 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. 15

17 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: Overlay 100 µl of 2X primary antibody cocktail solution on cells suspension (in Blocking/permeabilization buffer) and incubate for 1 hour at room temperature Centrifuge at 400g for 4 minutes and remove supernatant. Do not wash Add 100 µl of 1x secondary antibody cocktail solution and incubate for 1 hour at room temperature in the dark Centrifuge at 400g for 4 minutes, remove supernatant and wash once with 500 µl of incubation buffer 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. 16

18 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. 17

19 12. Troubleshooting Problem Cause Solution Low Signal High side scatter background Low event rate High event rate Signal not correctly compensated Insufficient secondary antibody Lasers not aligned Secondary antibody is not compatible with primaries Cells lysed Bacterial contamination Low number of cells Cells clumped High number of cells/ml Check positive single color control is set up correctly on flow cytometer and gated/compensated correctly to capture all events Increase concentration of antibody Run flow check beads and adjust alignment if necessary Use secondary goat antibodies raised against mouse and rabbit for this kit Ideally samples should be freshly prepared. Do not vortex or shake the sample at any stage. Do not exceed 500 x g for centrifugation Ensure sample is not contaminated Run 1x10 6 cells/ml Ensure a single cell suspension. Sieve clumps (30 µl nylon mesh) Dilute between 1x10 5 cells/ml and 1x10 6 cells/ml 18

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