I plan to work with EL-4 cell line, previously on culturing Jurkat e6.1 i found the cell were dying on sub-culturing. So i would need suggestions for optimum growth of EL-4. I have heard addition of BME helps. But what should be the amount that should be added in a 20 ml culture.
Also what stimulant should be used to activate its t cell response.
Is PMA(10 ng/ml) and inonomycin(500 ng/ml) a good stimulant?
Hi Sneha,
I culture EL-4 in the following medium:
RPME-1640, 10 % FBS, 1% Pen-Strep, 1% L-Gln, 1mM Na-pyruvate, 4.5g/l glucose, 0.05mM b-ME.
Stimulation depends on what do you want to investigate and what methods will you apply. I used 10 ng/ml PMA and 100 ng/ml ionomycin and hot nice gene inductions after 6h and 24h (IL-2, measured with qRT PCR).
Worth of noting that
”Original EL-4 cells were composed of the mixture of cells both with presence and absence of TCR/CD3 complex on their surfaces. The existence ratio of TCR/CD3 complex positive cells in original EL-4 cells was about 30%. They were also CD4+.”
( Kunugita N1, Mei N, Goncharova T, Norimura T. J Toxicol Sci. 2007 Oct;32(4):377-86.)
Zsuzsanna
I agree that Zsuzsanna's medium suggestion is generally suitable. In my hands, proliferation of EL4 cells is highly density dependent. Ideally, the culture should be maintained between 10e5 and 10e6 cells/mL. I am pretty sure that their growth is dependent on a secreted factor, because when I dilute the culture below 10e5 cells/mL, they stop dividing altogether (especially when using a recently-thawed batch).
Hi,
My cells were growing amazingly and i cryo preserved almost 5-6 million cells each time. However when I am thawing the cells after one spin i find some pellet, then by the next media wash (for removing traces of DMSO) the cell pellets are almost negligible. and on plating these cells they are barely growing.
How can i revive my cells?
Hi,
I have no difficulties getting them revived after freezing... For cryopreservation I use 5% DMSO in the normal culture media and freeze 4-8 million cells in 1 ml. When I thaw them I wash once with the culture medium and take them to a higher than normal density (about 0.5 - 1 million cells/ml) for a day and after that I dilute and subculture them in a density of 0.2 -0.3 million cells/ml.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line