I am now culturing RPMI8226 cell line and I had some problems after a week of culture. I observed some adherent cells that are growing "helthier" than those I observed in suspension (actually they seem to be burst).
I cultures these cell line in RPMI 1640 medium (Lonza) supplemented with 10% FBS in T25 flasks (Orange Scientific) and the cell viability was very low (30%). I also maintained some cells in 12 multi-well plate (BD Falcon) and reached higher viability rates (50-60%).
I cannot understand why they decreased cell viability suddenly (after a week of culture) since they are being cultured in the same manner.
Can you help me with this?
Thank you.
Hello
The cells culture in complete media (CM) consisting of RPMI 1640, 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin, and 10% fetal bovine serum FBS).
For Subculture and the cell concentration has reached 1 x 106 cells/ml. It is recommended to distribute the cells into new flasks containing fresh medium thus diminishing the amount of dead cells and cell debris . Adjust to a cell concentration of 1-2 x 105 cells/ml depending on the specification given for the cell line.
Look at paper here may find an explanation for your question
Good Luck
http://www.sciencedirect.com/science/article/pii/0385638087901397
http://cancerres.aacrjournals.org/content/50/6/1873.full.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917857/
Thanks Vanessa and Saleh.
I'll just start my research include culture RPMI8226 myeloma cell line
Beycan,
These cells are semi-adherent cells. To subculture, I used to retain the medium containing the free floating cells into a falcon tube and then I used trypsin only to detach the adherent ones (that were also tranferred to the same tube containing the free floating ones). I also kept the flasks in culture horizontally.
I hope this will help you, at least it worked for me.
Good luck,
Vanessa Pinto
Hi Vanessa,
I am culturing RPMI 8226 (with RPMI 1640, 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin, and 20% fetal bovine serum) and though ten days pass, we couldnt reach to enough concentration to freeze the cells. Can I learn the percent of tripsin in the solution that you use to detach the adherent cells ? I used firstly 24 well plate and now ı am culturing at T25 and putting incubator verically. By the way, ı couldnt remove the dead cells completely inspite of santrifuging at low rpm. Is there anything missing? Lastly, every 3 days, do you santrifuge the cells and culture at new flask or just add medium in the same flask?
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