7 replies to this topic

[Mamcy10]

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

Edited by Mamcy10, 21 October 2010 - 01:11 PM.

[bob1]

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.

[Mamcy10]

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

Mamcy

bob1, on 21 October 2010 - 03:08 PM, said:

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.

[leelee]

You could try adding some EDTA to your trypsin?

[Mamcy10]

Hi Veteran,

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

Mamcy

leelee, on 21 October 2010 - 06:30 PM, said:

You could try adding some EDTA to your trypsin?

[sushik]

bob1, on 21 October 2010 - 03:08 PM, said:

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

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.

Edited by sushik, 22 October 2010 - 05:28 PM.

[Mamcy10]

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 22 October 2010 - 05:25 PM, said:

bob1, on 21 October 2010 - 03:08 PM, said:

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

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.

[Mamcy10]

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 22 October 2010 - 07:25 PM, said:

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 22 October 2010 - 05:25 PM, said:

bob1, on 21 October 2010 - 03:08 PM, said:

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

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.