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Cell Surface Immunofluorescence Staining Protocol

互联网2013-11-13

4190

实验试剂

Cell Staining Buffer [ Details:BioLegend Cat. #420201]

Red Cell Lysis Buffer [ Details: BioLegend Cat. #420301]

7-AAD Viability Staining Solution[ Details: BioLegend Cat. #420403]

TruStain FcX™[ Details: anti-CD16/32, BioLegend Cat. #101319]

实验步骤

1. Harvest Tissue or Cells:

1) Obtain desired tissue (e.g. spleen, lymph node, thymus, bone marrow) and prepare a single cell suspension in Cell Staining Buffer (BioLegend Cat. #420201). If using in vitro stimulated cells, simply resuspend previously activated cultures in Cell Staining Buffer and proceed to Step 2.

2) Add Cell Staining Buffer up to ~15 ml and centrifuge at 350 x g for 5 minutes, discard supernatant.

2. Lyse Red Cells:

1) If necessary (e.g. spleen), dilute 10X Red Blood Cell (RBC) Lysis Buffer (BioLegend Cat. #420301) to 1X working concentration with DI water and resuspend pellet in 3 ml 1X RBC Lysis Buffer. Incubate on ice for 5 minutes.

2) Stop cell lysis by adding 10 ml Cell Staining Buffer to the tube. Centrifuge for 5 minutes at 350 x g and discard supernatant.

3) Repeat wash as in step 2.

4) Count viable cells and resuspend in Cell Staining Buffer at 5-10 x 10 cells/ml and distribute 100 μl/tube cells/tube) into 12 X 75 mm plastic tubes.

3. Block Fc-Receptors:

Reagents that block Fc receptors may be useful for reducing nonspecific immunofluorescent staining. In the mouse, purified anti-mouse CD16/CD32 antibody specific for Fcγ R III/II (BioLegend Cat. #101302, clone 93) can be used to block nonspecific staining of antibodies. In this case, block Fc receptors by pre-incubating cells with 5-10 μg/ml purified anti-CD16/32 on ice for 10 minutes. In the absence of an effective/available blocking antibody for human and/or rat Fc receptors, an alternative approach is to pre-block cells with excess irrelevant purified Ig from the same species and same isotype as the antibodies used for immunofluorescent staining.

4. Cell-Surface Staining with Antibody:

Add appropriately conjugated fluorescent, biotinylated, or purified primary antibodies at predetermined optimum concentrations (e.g. anti-CD3-FITC, anti-CD4-Biotin, and anti-CD8-APC) and incubate on ice for 15-20 minutes in the dark.

9) Wash 2X with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes.

1) If using a purified primary antibody, resuspend pellet in residual buffer and add previously determined optimum concentrations of anti-species immunoglobulin fluorochrome conjugated secondary antibody (e.g. FITC anti-mouse Ig) and incubate in the dark for 15-20 minutes. If using a biotinylated primary antibody, resuspend cell pellet in residual buffer and add previously determined optimum concentrations of fluorochrome conjugated Streptavidin (SAv) reagent (e.g. SAv-PE, BioLegend Cat. # 405204) and incubate on ice for 15-20 minutes in the dark.

2) Repeat step 9.

3) Resuspend cell pellet in 0.5 ml of Cell Staining Buffer and add 5 μl (0.25 μg)/million cells of 7-AAD Viability Staining Solution (BioLegend Cat. #420403) to exclude dead cells. Note, BioLegend does not recommend use of 7-AAD with either PE-Cy5 or PE-Cy7 antibody conjugates.

4) Incubate on ice for 3-5 minutes in the dark.

5) Analyze with a Flow Cytometer.

5. Immunofluorescent Staining of Whole Blood:

1) Add predetermined optimum concentrations of desired fluorochrome conjugated, biotinylated, or purified primary antibodies to 100 μl of anti-coagulated whole blood.

2) Incubate at room temperature for 15-20 minutes in the dark.

3) Dilute 10X Red Blood Cell (RBC) Lysis Buffer (BioLegend Cat. #420301) to 1X working concentration with DI water. Warm to room temperature prior to use. Add 2 ml of 1X RBC lysis solution to whole blood/antibody mixture. Incubate at room temperature for 10 minutes.

4) Centrifuge at 350 X g for 5 minutes, discard the supernatant.

5) Wash 1X with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes.

6) If using a purified primary antibody, resuspend pellet in residual buffer and add a previously determined optimum concentration of anti-species immunoglobulin fluorochrome conjugated secondary antibody (e.g. FITC anti-mouse Ig) and incubate in the dark for 15-20 minutes. If using a biotinylated primary antibody, resuspend cell pellet in residual buffer and determined optimum concentration of fluorochrome conjugated Streptavidin (SAv) PE, BioLegend Cat. # 405204) and incubate for 15-20 minutes in the dark.

7) Repeat step 5.

8) Resuspend cells in 0.5 ml Cell Staining Buffer or 0.5 ml 2% paraformaldehyde-PBS fixation buffer.

9) Analyze with a Flow Cytometer.

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