Measuring Proliferation using CFSE Dye

CFSE dye incorporates into the cell membrane, and upon subsequent cell divisions, the dye is split into daughter cells. For example, an activated cell line will have many divisions and show multiple peaks in the histogram analysis, compared to a non-activated cell line.


  1. Centrifuge cells of interest to obtain a cell pellet between 1x105 and 1x107 cells and aspirate supernatant.
  2. Gently resuspend cells in pre-warmed (37°C) proliferation dye such as CFSE at a predetermined concentration (perform a titration of CFSE to determine optimal concentration).
  3. Incubate cells for 15min at 37°C.
  4. Wash cells 2x in culture media by centrifuging cells (900 rpm for 3 min) and resuspending in pre-warmed media.
  5. Set up in vitro cultures under appropriate conditions (ex: tissue culture plates in a 37°C incubator).
  6. Harvest cells and stain for other surface markers if needed.
  7. Send samples to AVIVA for flow cytometry analysis using 488nm excitation and emission filters. Multiple peaks indicate cell proliferation.