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
- Dilute cells to 10x10^6 cells/mL
- Aliquot 100uL of cells per tube (1x10^6 cells total)
- 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.
- Add 10uL of appropriate diluted antibody to each tube (dilution is determined by antibody titration). Incubate for 20mins at 4C in the Dark.
- Wash 1x with FACS Buffer.
- Discard supernatant and re-suspend cells by gently flicking. Add 200uL FACS Buffer prior to analysis**.
- **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.
- Wash 2x with FACS Buffer, discard supernatant and re-suspend cells.
- If fixing cells for analysis at a later date, add .3ml to .4ml of 2% Formaldehyde to each tube and vortex.
- Store cells at 4 C in the Dark until analysis.
Use The following Template as a guide to set up Experiments: