Our lab recently did a test run mixed lymphocyte reaction (MLR) using normal human PBMCs.
Medium composition was HyClone RPMI-1640 +10%hiFBS +P/S +Fungizone +2.05 mM L-glutamine.
The feeder cells were treated with mitomycin C at 50ug/ml for 1 hour to render them non-proliferative.
DPBS was put in the space between wells to help minimize effect of evaporation and edge effects.
Cells were grown for 6 days at 5% CO2/100%RH/37'C. Incubator logs show no deviations.
Excess stock from which cells were taken was plated in a six-well plate in the same media and grown alongside the MLR plate. These cells are still alive. The 96-well plates used for MLR are brand-new. There was no sign of any bacterial, fungal or yeast contamination.
MLR is new to me, so it may be that I'm missing something obvious. What's going on here? Why are all my cells dead in the MLR but not in my 6-well plate?
Dear Carl,
Try mitomycin C 30ug/ml and keep me posted please.
Best
Mannan
- If you are not obtaining the cells form an infected site of a biopsy then remove fungizone.
- Be sure that you extensively wash MitoC away from the target cells before adding them into the MLRs.
- Prefer U-bottom 96-well plates and adjust the cell number accordingly.
- 4-day-long incubation can also work.
These are basic recommendations... you may also like to think about adding supplements like IL-2 or non-essential amino acids etc. to support cells' viability.
Thanks to both of you. I'm going to replate today using less mitomycin C and media with NEAA and without fungizone.
We always irradiate feeder cells, dose dependent on kind of feeder cell, instead of using MitoC. Might be an option to try if the equipment is avalable to you.
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