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IACF-Flow Cytometry Facility- Direct and Indirect Antibody Staining Protocol

Direct/Indirect Staining Protocol
Use this Protocol for Directly or Indirectly staining cells for Flow Cytometric Analysis

1) Dilute cells to 5x10^6 cells/mL

2) Aliquot 100uL of cells per per tube (5x10^5 cells total)

3) Add 20uL blocking reagent: 2.4G2 (anti-Fc receptor) for mouse cells, 10% goat, mouse, or rabbit serum for human cells. Incubate 5-10min at Room Temperature.

4) Add 10uL of appropriate diluted antibody to each tube (dilution is determined by antibody titration). Incubate for 20minutes at 4C in the Dark.

5) Wash 1x with Facs Buffer.

6) Discard supernatent and resuspend cells by gently flicking. Add 200uL Facs Buffer prior to analysis**.

7) **If primary antibody was not directly coupled, do not add 200uL FACS Buffer. Instead, add 10uL of appropriate secondary reagent. Incubate 20minutes at 4C in the Dark.

8) Wash 2x with FACS Buffer, discard supernatent and resuspend cells.

9) If fixing cells for analysis at a later date, Add 200uL Facs Buffer to each tube.

10) Add 50uL 10% Paraformaldehyde stock to each tube and vortex.

11) Store cells at 4 C in theDark until analysis.

Use The following Template as a guide to set up Experiments:

Cells.......................................... Date.....................................

Tube # Blocking Reagent Primary Reagent Secondary Reagent Tertiary Reagent Fixation
1 20ul 2.4G2       1% PFA
CD3 FITC    
CD19 Bio SA-FITC  
CD3 Pure Anti-mouse Bio SA-FITC