Extracellular Cell staining

Developed by: Justin Meyers

Sample: Cell line or primary cells

Materials

• Directly conjugated antibodies
• FACS buffer (DPBS + 5% FBS) 
• BD Cytofix (BD 554655) or 1% paraformaldehyde

Procedure

Isolate cells

1. Harvest cells, want good single cell suspension
2. Count cells
3. Dispense aliquots of 1 million cells into labeled tubes
4. Add 5 ml FACS Buffer and centrifuge for 5 min at 1300 RPM
5. Re-suspend cell pellet in 100ul FACS Buffer with gentle vortex

Fix cells:

1. Add appropriate amount of antibody
2. Vortex gently
3. Incubate at 4C for 30 min
4. Add 4 ml of FACS Buffer
5. Pellet cells at 1300 rpm for 5 min
6. Re-suspend cells in 500 ul of FACS Buffer
7. Keep cells on ice up to 1 hour before analysis*
8. Analyze cells on flow cytometer

*If cells cannot be analyzed within one hour, fix the cells in 0.5 ml of 1X BD Cytofix or 1% paraformaldehyde and store at 4C for analysis the following day