Intracellular Staining for Ki67

 

  • Surface stain 3 x 10^6* cells in a v-bottom tube for 15 min @ 4°C in the dark.
  • Wash with FACS buffer and resuspend cells in 50ul cold PBS.
  • Fix cells by adding 500ul 0.5% PFA drop-wise down side of tube while gently agitating cells on the vortex. Incubate for 30 min at RT in the dark.
  • Wash** and permeabilise cells by adding 500ul 1x FACS Permeabilsing Solution (BD) drop-wise down side of tube while gently agitating cells on the vortex. Incubate for 30 min at RT in the dark.
  • Wash and add 20ul anti-human Ki67 or isotype control to cells. Incubate for 30 min at RT in the dark.
  • Wash and resuspend in 200ul FACS buffer.

* - When analysing minute populations, surface stain twice as many cells with 60 m l of antibody then pool sample and proceed as per usual (since subsequent steps are added in excess).

** - For all washes after step 3, recover cells by centrifugation at 1800 rpm for 5 min to better pellet ‘fluffy’cells.