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 10x10^6 cells/mL

  1. Aliquot 100uL of cells per tube (1x10^6 cells total)

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

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

  1. Wash 1x with FACS Buffer.

  1. Discard supernatant and re-suspend cells by gently flicking. Add 200uL FACS Buffer prior to analysis**.

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

  1. Wash 2x with FACS Buffer, discard supernatant and re-suspend cells.

  1. If fixing cells for analysis at a later date, add .3ml to .4ml of 2% Formaldehyde to each tube and vortex.

  1. Store cells at 4 C in the Dark until analysis.

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

Tube #

Blocking Reagent

Primary Reagent

Secondary Reagent

Tertiary Reagent

Fixation

1

20uL 2.4G2

2% FA

2

20uL 2.4G2

CD3-FITC

2% FA

3

20uL 2.4G2

CD4-FITC

CD8-PE

2% FA

4

20uL 2.4G2

CD19-Bio

Secondary Ab-FITC

2% FA

5

20uL 2.4G2

CD3-Pure

Anti-mouse Bio

Secondary Ab-FITC

2% PFA