Extracellular Cell staining

Developed by: Justin Meyers

Sample: Cell line or primary cells


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


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


Jill Hutchcroft
Flow Cytometry Facility Director
Phone: (765) 586-1130
Email: hutchcroft@purdue.edu