We typically use 0.5 - 1 × 106 cells in a 50-100 μl experimental sample (a test). Since applications vary, each investigator should titrate the reagent to obtain optimal results.
Cells should be rinsed with PBS to remove serum proteins prior to antibody staining. If staining with more than one antibody, prepare a pool of antibodies together.
All incubations should be performed on ice and with minimal light exposure.
Antibody Staining Protocol
Fc Block of Cells from Tissues
1. Phagocytic cells (such as macrophages and granulocytes) can bind non-specifically to antibodies unless the Fc receptors of these cells are blocked. If the sample is from a tissue homogenization possibly containing macrophages, or from a cell culture line expressing Fc receptors (such as Daudi and THP-1), you can use anti-Fc Receptor non-labeled antibody (anti-CD16/32 in mice OR human IgG in human) to block the receptors. Samples of cells without Fc Receptors can skip the Fc Block step.
2. Add 1 μg of Fc Block Antibody directly OR 5 μg human IgG to 100 μl of suspended cells.
3. Incubate on ice for 20 minutes.
4. Proceed directly to Primary Antibody Staining (no need to rinse away Fc Block Antibody). Adaptable for fluorescent primary antibody, not for Secondary Antibody Staining.
Cell Surface Staining of Human PBMCs and Cell Lines
Primary Antibody Staining
1. Add 1 μg of primary antibody directly to 50-100 μl of suspended cells.
2. Incubate on ice for 20 minutes.
3. Add 1 ml PBS to rinse non-bound antibody.
4. Centrifuge at 1200-1500 rpm for 5 minutes. Repeated this step once again. Decant and resuspend cells in residual fluid (~100 μl). If your primary antibody is labeled with a flurorchrome, skip to step 10.
Secondary Antibody Staining
5. Add 1 μg of labeled appropriate secondary antibody directly to the 50-100 μl of rinsed cells.
6. Incubate on ice for 20 minutes.
7. Add 1 ml PBS to rinse non-bound antibody.
8. Centrifuge at1200-1500 rpm for 5 minutes. Repeated this step once again.
9. After centrifugation, decant the supernatant from the cell pellet. Resuspend the cells in residual fluid.
Dilute cells and analyze
10. Add 200-400 μl of PBS to dilute cells. Check the sample for cell clumping. Cells that clump must be filtered through a 70 μ mesh filter prior to Flow cytometry to prevent clogging of the instrument. The sample is ready for Flow analysis.
Fixation of cells
If you will not be able to analyze the cells by Flow Cytometer within a few hours (usually 4-6 hrs) of staining, you should fix the cells.
11. Prepare a solution of 1% Paraformaldehyde in PBS.
12. After resuspending cells in residual fluid (when staining is complete at step 4 or step 9), add 250 μl 1% Paraformaldehyde solution.
13. The sample should be stored in the dark in the refridgerator and can be read at least several days later.
14. Prior to submitting the sample for analysis, check for cell clumping.
Fixation may quench some fluorescent proteins such as GFP.
Be sure to schedule your Flow Analysis at least 24 hours in advance.
Be sure to schedule your Flow Sorting at least 1 week in advance.
1. Appropriate negative controls should be used to verify specificity and rule out background staining. An irrelevant antibody of the same isotype and concentration should be tested to aid in setting quadrant statistics.
2. Fixation of cells using extremely harsh conditions or for a prolonged period of time may alter the target epitope and render it unrecognizable to the antibody. Strict attention to the recommended fixation conditions must be followed in order to generate consistent results.
3. Resuspend a maximum of 0.5-1×106 washed (or surface stained) cells in 0.25 mL of Fixation/ Permeabilization solution and incubate on ice for 30 minutes.
4. Vortex the cells intermittently in order to maintain a single cell suspension. Following fixation wash the cells twice in 1×Perm/ Wash buffer by centrifugation at 1500 rpm for 5 minutes.
5. Decant the supernatant ensuring that approximately 50-100 μL of Perm/ Wash buffer remains in the tube.
6. Gently resuspend the cells in the remaining Perm/Wash buffer and add 1 μg(or a previously titrated amount) of antibody conjugate.
7. Briefly vortex the tube and incubate for 30 minutes on ice in the dark.
8. Wash the cells twice using 1 mL of Perm/Wash buffer each time. Centrifuge as in step 9.
9. Resuspend the cells in each tube with 200-400 μL of PBS OR 1% Paraformaldehyde solutionfor analysis by flow cytometry.