I'm trying to stimulate frozen dog's PBMC in culture ( I used conA and PMA/Ionomycin) and monitor the proliferation with CFSE. I'm having some troubles with the quality of the CFSE peaks and the cell viability:
-with 1x10^6 cells/well they had a good viability even after 6 days, but I couldn't obtain nice CFSE peaks, I had more like one big large peak who decreased in fluorescence intensity during the days.
-I tried with less cells pro well (1x10^5cells/well), same conditions and they were dead after 3 days...
since I would like to work with as few cells as possible, I'm planning to try again with for ex. 5*10^5 cells/well, but I still have to fix the problem with my CFSE peaks.
I also noticed that the cells downregulate CD4+ after some days, which makes it difficult to identify the lymphocytes in the population. I was also wondering to introduce another lymphocytes surface marker...
Some suggestions?
Hi Franco,
you could try to lower the CFSE concentration used to label your cells to obtain separate peaks. Stain your cells for CD3 to identify T cells.
success!
I have no experience with frozen canine PBMC but I did have a similar problem with the 'CFSE' (we use Celltrace Violet) labelling of fresh canine PBMC. Directly after the labelling with CFSE, try including some additional washing steps with ice-cold sterile PBS with a protein source (e.g. 1% BSA or 1% FCS) before putting the cells in culture. If labelled with 5 µM and stimulated with 1.25 µg/ml ConA, you should see 5-7 distinguishable peaks in your flow cytometry results after 4 days incubation.
Are you resting your cells overnight after thawing and before labeling? This may help with viability.
Many thanks to everyone for your answers!
@Bert: at the beginning I used CFSE at a final concentration of 2.5uM. I then reduced it already to 1uM. I will definitely try to lower it even more if I'll still have this issue.
@Michael: after the staining I wash my cells with 100%FCS and then 2 more times with PBS(10%FCS). Like I said after a titration I label them with 1uM CFSE and try to stimulate with 1ug/ml or 2ug/ml conA, but I still wasn't able to see nice peaks. I don't know if the staining protocol I'm using isn't so good for frozen cells.
@Colt: No I'm not resting them! This may be a good idea.
- Badges
- Science method