Hi everyone,
I am facing HUGE difficulty in growing Ramos (Human Burkitt's lymphoma B cells).
Here's my media recipe:
RPMI 1640
1x Glutamax (100x Glutamax stock from Thermofisher)
10000U PenStrep Antibiotics
20% FCS
My resuscitation and culture methods:
I resuscitated Ramos cells (previously stored down at 10% DMSO, 90% FCS) from liquid nitrogen storage and thawed aliquots in 37˚C water bath before diluting out the DMSO with fresh media. I then centrifuged the cells at 1500rpm for 5 mins, removing the media completely after. Resuspended with 5mL of fresh media and did a cell count. The counts I got was approximately 6x10^5 cells/mL, 93% viable cells. I then topped up with 5mL of fresh media and cultured the cells in T25 tissue culture flask (placed upright). I also changed the media next day to make sure that no residual DMSO was left in the culture.
Two days later, I did a cell count and found that I got 97% viable cells at 1.2x10^6 cells/mL in T25 flask. I decided to split the culture (did a 1 in 2 dilutions- Added 10mL of fresh media to 10mL of existing culture) and transferred the entire 20mL of culture into a T75 flask. The next day when I counted the cells again, they were dead (~approx 90% cell death, confirmed with Trypan blue staining). Growing 10% viable population was tough even in 24 well plate. I ended up having to resuscitate another vial from the liquid nitrogen storage tank.
A shoutout to the experts using Ramos cells in their research projects, do you encounter similar issues? Do you have a successful protocol for culturing Ramos? Also, if you have identified any possible mistakes in my culture techniques, could you please let me know?
Your attention to this post is deeply appreciated.
Kind regards,
Ferrer
Just a quick answer:
On the page of ATCC they give culture medium and also great documentation to help you about this subject:
https://www.lgcstandards-atcc.org/Products/All/CRL-1596.aspx?geo_country=ch#documentation
You can check with the publications in here and also with the recommandations from ATCC for the culture.
Regards,
Mathieu
Thanks Mathieu,
I started culturing the cells using ATCC's protocol but they did not resuscitate well. Which was why I decided to increase the % FCS in the media. Surprisingly, they were happy with it for a short period of time. Thank you for your suggestion and I will look more closely into other publications.
Kind regards,
Ferrer
Hi Ferrer Ong,
This protocol is online, but it works well for this cell line.
Raji (ATCC number CCL-86): Burkitt’s Lymphoma; B lymphocyte cell line Growth medium: RPMI Mediujm 1640 (GIBCO # 21870) + 10% FBS + 2mM L- Glutamine + 100 units/ml penicillin + 100 micro-g/ml streptomycin (GIBCO # 15140-122).
Protocol for Thawing Raji Cells:
1. Take out the Raji stock vial from liquid nitrogen (we freeze at 1x10^6 cells per vial) and thaw it in 37° water bath. Suspend thawed cells in 5 ml growth media. 2. Centrifuge at 1000 rpm for 3 min, discard media.
3. Resuspend cell pellet in 15ml growth media and transfer cells into a 75 sq. cm. tissue culture flask. Cells are grown in a 37oC incubator at 5% CO2.
Protocol for Subculturing of Raji Cells:
Change medium every 2 to 3 days, and split cultures when they reach 2-3x10^6 viable cells/ml.
Transfercellsuspension(ingrowthmedium)tocentrifugetubesandspin at 1000rpm for 3minutes.
Aspiratesupernatantandadd10mlsoffreshgrowthmedia
Countcells,andtransfer0.5-5X10^5viablecells/mltoanewculture
vessel (this can either be a spinner flask or a tissue culture flask that’s placed horizontally a 37oC incubator at 5% CO2.)
Note: We typically split cells at a ratio of 1:10 every 2 to 3 days using the abovementioned seeding conditions (and we grow a back-up maintenance plate, in addition to the culturing vessel used for experiments, by adding about 1x10^5 cells per 75 sq. cm flask).
Freezing of Raji Cells:
Cells can be stored as a stock in liquid nitrogen at 1x10^6 cells/ml in growth medium containing 10% DMSO.
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