I have tried a histopaque isolation, but I get a lot of RBCs in my final pellet.
When I later put these cells on a 7 day CFSE assay with PHA 0.5ug/ml as a positive control, I find two problems.
1- All the cells are dead.
2- There are so many RBCs it is difficult to find the CBMC population in the FACs.
Now, I culture in RPMI+ P/S 2%, + HEPES 2%, +l-GLUT 1%, +10% FCS.
For CFSE, i USE 0.125mM per 1x10^6
Does anyone have any suggestions that might help?
Definitely decrease the concentration of CFSE you use - I use it with mesenchymal stem cells at 2microM, higher concentrations killed them. Lower concentrations were not allowing long-term culture/analysis as the cells were dividing to much and losing staining.
Hope this helps
C.
For labeling human adipose derived MSCs with CFSE we saw toxicity at 5 microM. We use CFSE at 1 microM to successfully label the cells. They are a little dimmer under fluorescent microscopy, but are still extremely bright by flow cytometry.
As for your isolation issues, try using a RBC lysis buffer (like ACK lysis buffer) after isolation. It will rupture the RBCs, but other cells tolerate it for a few minutes. Also, if you are getting a lot of RBC after histopaque, you may need to adjust the dilution to make sure the density is correct. You may also try the Accuspin system, which uses a frit in the tube to aid in separation.
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