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
2	"	CD3 FITC			"
3	"	CD4 FITC	CD8 PE		"
4	"	CD19 Bio	SA-FITC		"
5	"	CD3 Pure	Anti-mouse Bio	SA-FITC	"