hi every one, i want to isolate antigen-specific CTL. my plan is as follows:
1. immunize mouse with peptide twice, at 2 weeks interval;
2. after 2weeks of second immunization, kill mouse and isolate spleen;
3. smashes spleen to make single cell suspentioan ,adjust cell density to 107/ml and
then cultivate for 6-7day;
4. finally i want to isolate live lymphocytes from these cultured cells,
so my question is :
a. after in vitro cultivation, can i use percoll or ficoll to distinguish live and dead cells?
b. if i can use percoll or ficoll isolate my desired cells, then how to do that?
i really need some help, thanks very much!
1) Percoll or Ficoll are used to separate out cells based on their weights/densities. Because lymphocytes are differently sized compared to other splenocytes, you will be able to use percoll/ficoll (diluted at the correct density) in order to isolate them. Refer to this data sheet to figure that out: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/p4937pis.pdf
2) Percoll does not distinguish live from dead. Depending on your downstream application, that might help. For example: if you were doing flow cytometry, you can add a viability dye which will allow you to gate only live cells. If you are culturing cells for 6-7 days, however, and you know that the culture method is effective, there is no reason why there should be a lot of dead lymphocytes. Make sure you have appropriate stimuli and media being used.
