Hi everyone, I got the two cell lines from DSMZ and wonder a bit about the culture conditions. I want to use the conditioned medium from WEHI3 for the BA/F3 cells. DSMZ recommend the usage of b-Mercaptoethanol in the medium. Is it really necessary? And what cell density of the WEHI-3B is good for the harvest of the conditioned medium? Can I freeze aliquots of the conditions medium for later supplementing the BA/F3?
Thank you for your advise!
Dear Lena,
BA/F3 cells are not picky at all. I have never used beta-mercaptoethanol, it is probably to keep the IL7R from forming aggregates. The only trick to keep in mind is that you add IL3 or IL7, otherwise the cells become IL-independent within a week or two. Do not grow to much more than 1.5 million/ml. They grow fast :-)
Dear Dirk,
thank you for your answer. I would like to use the supernatant from WEHI3 to supply IL3 instead of buying the expensive cytokines. Do you used them as well? For how long do I have to condition the medium by WEHI3? And do you think I can freeze the medium to use as a supplement later?
Best, Lena
Hi Lena,
We never used supernatant for BA/F3, because we wanted to be sure which cytokine we added to the BA/F3 cells for its growth. But we were looking at cytokine signaling, so we were perhaps more picky than you have to be.
Conditioning takes place in 2-3 days, when your WEHI3 cells grow from 0.3-0.5 to 1.2-1.7 million cells per ml. Do not let the cells grow denser than that. It is possible to freeze the medium, but make sure you do not thaw batches more than 1-2 times. I cannot stress enough how important it is to filter the medium through a 0.22 micrometer filter before storage or use. Conditioned medium is famous for its contamination problems.
Good l;uck!
D.
Dear Dirk,
thanks again for your quick answer. The WEHI-3B are doing well in culture, so I can start to collect the supernatant soon and thaw the BA/F3. Indeed I am not so into the cytokines itself, because I want to study the influence of transcription factor overexpression. So, I keep my fingers crossed, that the conditioned medium is good enough!
Best Lena
HI! I add 10ng/ml IL-3 to the media, but my cells are not growing well. Do I have to add fresh IL-3 to the media everyday? Also, are the cells very small compared to SNU cell lines or 293T cells?
Hi Keira,
you mean the BA/F3 cells? Yes, they are tiny, suspension cells. But mine where growing very good! But we never added IL3 directly, we where using the WEHI supernatant. Since we didn't changed the medium daily, I would expect that the IL3 will stay stable for several of days.
Thank you! That makes me feel better. Yes, the ba/f3 cells, I've never dealt with cells this small! Okay will monitor then, I was worried they will be high maintenance...thank you!!
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