BioLegend_Surface_Staining_Flow_Protocol_060215

Cell Surface Immunofluorescence Staining Protocol
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. No. 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.
Lyse Red Cells:

3. If necessary (e.g. spleen), dilute 10X Red Blood Cell (RBC) Lysis Buffer (BioLegend Cat. No. 420301) to 1X
working concentration with DI water and resuspend pellet in 3 ml 1X RBC Lysis Buffer. Incubate on ice
for 5 minutes.
4. Stop cell lysis by adding 10 ml Cell Staining Buffer to the tube. Centrifuge for 5 minutes at 350 x g and
discard supernatant.
5. Repeat wash as in step 2.
6. Count viable cells and resuspend in Cell Staining Buffer at 5-10 x 106 cells/ml and distribute 100 μl/tube of
cell suspension (5-10 x 105 cells/tube) into 12 x 75 mm plastic tubes.
Block Fc-Receptors:
7. Reagents that block Fc receptors may be useful for reducing nonspecific immunofluorescent staining. In
the mouse, TruStain fcX™ (anti-mouse CD16/32) Antibody specific for FcγR III/II (BioLegend Cat. No.
101319, clone 93) can be used to block nonspecific staining of antibodies. In this case, block Fc receptors
by pre-incubating cells with 1.0 μg of TruStain fcX™ (anti-mouse CD16/32) Antibody per 106 cells in a
100 μl volume for 5-10 minutes on ice. In humans, cells can be pre-incubated with 5 μl of Human TruStain
FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301) per 100 μl of cell suspension for 5-10
minutes at room temperature. In the absence of an effective/available blocking antibody, 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.
Cell-Surface Staining with Antibody:

8. 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.
BioLegend | San Diego, CA 92121

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Rev. 060215
10. 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 on ice 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. No. 405204) and incubate on ice for 15-20 minutes in the dark.
11. Repeat step 9.
12. 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. No. 420403) to exclude dead cells. Note, BioLegend does
not recommend use of 7-AAD with either PE-Cy5 or PE-Cy7 antibody conjugates. For cells that must
be fixed prior to staining, BioLegend recommends Zombie Live/Dead Fixable Dyes.
13. Incubate on ice for 3-5 minutes in the dark.
14. Analyze with a Flow Cytometer.
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. No. 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 add a previously
determined optimum concentration of fluorochrome conjugated Streptavidin (SAv) reagent (e.g. SAv-PE,
BioLegend Cat. No. 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.

Key Reference: Current Protocols in Cytometry (John Wiley & Sons, New York), Unit 6 Phenotypic Analysis.
Reagent List:
Cell Staining Buffer (BioLegend Cat. No. 420201)
Red Cell Lysis Buffer (BioLegend Cat. No. 420301)
7-AAD Viability Staining Solution (BioLegend Cat. No. 420403)
TruStain FcX™ (anti-CD16/32, BioLegend Cat. No. 101319)
Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301)


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Cell Surface Immunofluorescence Staining Protocol

Harvest Tissue or Cells:

Lyse Red Cells:

Cell-Surface Staining with Antibody:

BioLegend | San Diego, CA 92121

Immunofluorescent Staining of Whole Blood:

BioLegend | San Diego, CA 92121

BioLegend | San Diego, CA 92121

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