Surface Staining for FACS

 

  1. Wet 2 glass slides with FACS Buffer and gently disrupt freshly dissected organ between frosted sides
  2. Recover cells by centrifugation at 1500rpm at 4ºC for 5min
  3. For Spleen cell suspension first nick the organ with scissors a few times to assist suspending
  4. For spleen and bone marrow cells resuspend in RBC Lysis buffer, (1mL for 1 min) after suspending, then wash with FACS Buffer
  5. Use the Coulter Counter (10microlitres suspension in 10mL Coulter Cup) to determine cell count
  6. Surface stain 3x10^6 cells with 30uL of titrated antibody in a 96 well U bottom plate for 20 mins @ 4ºC
  7. Wash by adding 170uL FACS buffer and recover cells by centrifugation at 1300rpm for 3mins. Wash twice if antibody is biotinylated
  8. If primary antibody is biotinylated, wash twice then repeat steps 6-7 for secondary antibody
  9. Resuspend in 200µL FACS buffer for acquisition

** When analysing minute populations, surface stain twice as many cells with 60uL of antibody (in two wells) then pool sample for acquisition