I am having trouble with my cultures of LCLs. After thawing, the cells are ok and they proliferate for 1-2 days. But after this initial proliferation the cells start to die and don´t recover. The medium doesn't become acidic, which I think is one of the reasons it is not growing well. I am always maintaining the cells around 5-10 x10e5/mL, in a 25cm2 flask standing upright. I am using RPMI medium (Sigma r8556) suplemented with HEPES, antibiotics, L-GLU, sodium bicarbonate and 10% FBS. I have used this medium before with these cells and did not have a problem. In the same incubator I am using other cells that are very sensitive and are growing well, like primary T cells (with the same medium).
It can happen that LCLs are slowly growing after thawing. Use only 3ml medium after thawing, use insulin (endconcentration about 5ug/ml, e.g. Roche cat.no 11376497001).
Next idea: If the pH of the medium does not change I would check the CO2 of your incubator. The CO2 content depends on huminity - maybe the water reservoir of your incubator is empty. ;)
I simply culture them in RPMI/10%FCS in an incubator at 37 degrees/5% CO2. Don't split them too early, I usually wait until media turns yellow. Then I split them every 3-4 days 1:3 to 1:5.
I agree with Magdalena Hagn except using 15% FCS. Don't split too fast unless medium get yellow.
Magdalena is right. Keep the volume low initially, cell density highish (2-10x10e5/ml viable cells), and you should see the cells start to clump up - this is a good sign. Down the microscope (in culture), LCLs will that are healthy will appear shiny, and often have spiky protruberances.
Basic media is fine (we use RPMI.10%FCS/L-glutamine/P&S) though serum batch can be important. Also bear in mind that LCLs can go through growth crises (typically one at 4-6 weeks post infection and another at 3-6 months). They may struggle at these times, and may or may not survive.
- Badges
- Science topic