MDA-MB-231 cell culture issue - after thawing first passage is fine, next goes bad (Oct/21/2010 )

Dear friends.

I badly need your help. I thawed MDA cells (P # 4) and cells looked fine in the T75 flask. I then subcultured after 80% confluency by trypsinization but even after keeping for 15min, all the cells did not come out. I slightly tapped the flask and also scraped the cells to get them out. I plates the cells that were detached and when they grew, half of these seemed to be necrotic. The media I am using is DMEM (high glucose) + FBS (10%) with NEAA, L glutamine and antibiotics. Last time I thawed I had same problem (I did not scratch cells in this one)but my very first stock (that I had purchased) was fine until P # 20 with same media. I am freezing in 70% DMEM + 20% FBS+ 10% DMSO (stepwise freezing).

Please help.

Thanks
MK

Did you wash the cells in PBS before adding trypsin?

Scraping cells is not good for keeping them viable. Avoid this proceedure if you want to have healthy cells.

Hi Bob,
Thanks for the reply.I washed with HBSS before trypsinization.

Mamcy

bob1 on Thu Oct 21 23:08:04 2010 said:

You could try adding some EDTA to your trypsin?

Hi Veteran,

This is Trypsin-EDTA 0.5% that I am using. What else cam I do?

Mamcy

leelee on Fri Oct 22 02:30:17 2010 said:

bob1 on Thu Oct 21 23:08:04 2010 said:

I agree, scraping cells is never good for cell viability and I've never had to do that with MDA-MB-231s.
All the recipes I've seen for HBSS have calcium and magnesium. I always do my pre-trypsinizing washes with PBS that does not have Ca++ or Mg++.

One thing I find to be helpful while trypsinizing is to keep the cells in the 37C incubator and check it periodically. Usually my MDA-MB-231s are quick to detach using 0.25% trypsin/EDTA (Gibco) with more than 90% floating after 5 minutes.

If you still have trouble, I've used Accutase to remove the most difficult cell lines (macrophages in particular) without having to scrape.

Hi Sushik,

Thanks for your reply. I use HBSS (invitrogen) with no Ca and Mg. The problem comes with T75 flask. With petri plates it takes 5 min for cells to detach. I do keep in incubator for 10-15min but get only 30% cells out in T75 flask. I will try using the other reagent you mentioned.

Thanks
Mamcy

sushik on Sat Oct 23 01:25:49 2010 said:

Hi guys

I checked my HBSS again. It does have Ca and Mg so I tried PBS wash and the cells did come out nicely and now they are good. The culture with bad cells was thrown. I think their problem was that I seeded at a very low density and partly scraping can be another reason.

Anyway ..thanks a lot guys..
Mamcy

Mamcy10 on Sat Oct 23 03:25:44 2010 said: