I have isolated some mouse splenocytes by meshing the spleens through cell strainers (70um). However due to bad timing I have to wait for at least 2 days for the reagents to come. I wonder if I can culture splenocytes instead of putting them in the fridge for 2 long days??
In the isolation protocol DMEM with 10%FBS is used to suspend splenocytes in the last step. Can I just culture them in DMEM-10 for a couple of days??
I also see that using RPMI with 10%FBS can keep splenocytes alive. Which one is better or are there any good cocktails for mouse splenocyte culture?
It would depend on the cell type you are trying to study. Having isolated cells from mice spleens, i can tell you that 2 days can change a lot of things, so much so that the experimental outcome can be completely different. We basically isolated the cells, tagged them with antibodies, and sorted a specific population of the cells like, gr1+, cb11b+, cells for example, and isolated the mRNA all in the same day. Any time a step was delayed, there was a huge difference in the number of cells collected, and in the population of the cells we were looking for.
If the reagents needed for the experiment is cheap, and doesn't take much time, I would try it by culturing in rpmi with 10% fbs. But it may be just better to start over as the results won't really be usable, i suspect.
Thanks so much for everyone's suggestions.
I was planning to sort out CD45+ cells from mouse spleen for further treatment.
I have actually done a trial before asking this question. I 'cultured' the cells harvested from mouse spleen (RBC removed) for 1 week in DMEM 10% FBS. I found that there were many suspension cells, not the adhesive fibroblasts, having a round shape like a healthy cell. I could extract RNA and protein from the suspension cells, but sorting with CD45 only yield
RPMI media is better than DMEM and as I know spleen can not be store, so if you want to do experiment using spleen cells, yafter isolation you have to use it directly for better results~
- Badges
- Science topic