I have been trying to replicate some CFSE proliferation data that I have performed in the past. I have been getting some strange results, there has been no sign that cells have proliferated when I run them on the flow cytometer (MACSQuant 16). The standard peaks we expect to see are absent and I either get one population CFSE+ or there is a large CFSE- population with hardly any CFSE+ still in the sample. In both examples when cells are counted on the Nexcelom counter there are many more cells after activation than what went into the flasks/wells. So that is my first issue, I'm wondering if it has to do with how the MACSQuant voltages are set, possibly? I've tried titrating up to double and down to 1/10th the recommended concentration of CFSE but with the same results.

The second issue is that when Samples are run day of staining there are two positive populations present with different median intensities. I've attached the gating strategy I used most recently on isolated mouse splenocytes (Pan-T isolation) but the results have been the same even when staining MV-4-11 cells from culture. Could this be some issue that I'm not aware of with the CFSE Stain itself?

Protocol is as follows and pulled more or less directly from the BioLegend data sheet:

1. Prepare a 5µM working solution by diluting 2µL of 5 mM CFSE stock solution in 1998µL 1X DPBS.

2. Resuspend cells at 1 e7 cells/mL in the CFSE working solutions. Resuspend the unstained in 1X DPBS.

· Unstained: 1.0 e7 cells in 1000µL of 1X DPBS

· 5µM CFSE: 2 e7 cells in 2000µL of CFSE Staining Solution

3. Incubate cells for 20 minutes at 37°C and keep protected from light.

4. Quench the stain with cRPMI by adding 5 times the original staining volume.

· Unstained: 5mL cRPMI (10% Heat Inactivated FBS + 90% RPMI + Pen/Strep)

· 5µM CFSE: 10mL cRPMI (10% Heat Inactivated FBS + 90% RPMI + Pen/Strep)

5. Centrifuge cells at 500 x g for 5 minutes and aspirate supernatant.

6. Resuspend in 1x106 cells/mL pre-warmed cRPMI-1640 medium.

· Unstained: 1.0 e7cells in 10mL cRPMI

· 5µM CFSE: 2 e7cells in 20mL cRPMI

7. Incubate cells at 5% CO2 at 37°C for 10 minutes.

8. Centrifuge cells at 500x g for 5 minutes and aspirate supernatant.

9. Resuspend 1e6 cells/mL in pre-warmed fRPMI-1640 medium and add cells to flasks according to table.[ALdM1] [NL2]

· Unstained: 1e7 cells in 10mL cRPMI

· 5µM CFSE: 2 e7 cells in 20mL cRPMI

11. Stimulate the cells by spiking in anti-mCD28 at 5µg/mL (50µL/mL) of the total volume in the flask.

· Unstimulated wells will receive fRPMI alone.

12. Gently swirl flask to mix.

3. Incubate cells at 5% CO2 at 37°C for 6 days standing vertically.

14. On day 3 gently pipette the culture up and down to break up cell clumps.

15. Every two days get a count and split the cell culture 1:2 with fresh fRPMI as necessary.