I have froze PMBCs in Complete D-MEM. Should I be thawing these samples in the same media as I froze them or switching to RPMI for the thawing process?
L-glutamine is present in RPMI but not in DMEM. Sodium pyruvate is present in DMEM but not RPMI.
Would this make a difference to the viability of my cells?
Hi Ganisha,
i would use the same medium to thaw the cells. You can add L.-glutamine to your DMEM. Next time I would use FCS+10%DMSO to freeze the cells.
Karsten
Hi, I agree with Karsten
When you freeze the PBMC you should have 10% DMSO inside, DMSO avoid damage to the cell during freezing. So I`m not shore if your cells are still alive, so after thaving check the viability.
You can freez cells in medium containing 10% DMSO or FCS containing 10% DMSO.
The DMSO is important to avoid cristal formation during freezing (see answer of Natasha). The cristals will damage the cells. However, DMSO (in high concentrations) is toxic to cells if it is metabolized (during thawing). Thus, I would use use 10% DMSO instead of 20% DMSO. You can use medium (+DMSO) or FCS (+DMSO). Both should work. However, In my hands FCS+10%DMSO works best.
To freez cells spin them down and resuspend in cold FCS/Medium + 10%FCS. Freez cells first 24h at -80°C and tranfer them later to liquid nitrogen.
For thawing be fast: Thaw the cells and transfer them imedeately to 50ml cold medium and wash them (to remove DMSO). For most cell types 1x washing is O.K. You have to avoid the toxic effects of DMSO.
I hope it will work,
Karsten
- Badges
- Science topic
- Similar topics
- Social Science
- Media