5 replies to this topic

[Research Panda]

Hi Everyone,

I'm new to immunology   and now culturing NIH3T3-CD40L for proliferating B cells.

However, I meet serious trouble in culturing NIH3T3-CD40L cells as I found that the NIH3T3-CD40L cells detached from 6-well plate (TPP) seriously after seeding B cells (PBMC) for 2 to 3 days.

I also observe that there is a change in the growth rate of the NIH3T3-CD40 cells. At first, I seed 0.4 x 10^6 NIH3T3-CD40L cells to a well and it can confluent on the next day. However, it took two days to confluent after several passage of NIH3T3-CD40L cells. I seed 2 to 3 x 10^6 cells in one T75 flask and took two days to confluent. I wonder if there is anything wrong during the passage of 3T3 cells??

Is there any tricks or tips in culturing NIH3T3-CD40L cells? Will the use of Corning 6-well plate better than TPP 6-well plates?

Please kindly help!

THANK YOU VERY MUCH!!!

[bob1]

Types of plate is unlikely to make much difference, it could be that you are sub-culturing your cells after they have reached a high density so the growth has slowed or you could have a contamination such as mycoplasma affecting your cells - get them checked.

[Research Panda]

bob1, on Aug 11 2009, 09:22 AM, said:

Types of plate is unlikely to make much difference, it could be that you are sub-culturing your cells after they have reached a high density so the growth has slowed or you could have a contamination such as mycoplasma affecting your cells - get them checked.

Thank you very much Bob! I'm trying to thaw a new cell line of 3T3 and lowering the density. Is there any optimal density for culturing 3T3? In addition, how can mycoplasm be checked? I heard from other lab that mycoplasma contamination is difficult to be detected.

Many thanks!!  

[bob1]

There are many threads on contamination in the cell biology sub-forum.  Basically it boils down to use the immunofluorescence method and it is best to send them away to an experienced company (which is quite expensive, but worth it).

[Research Panda]

bob1, on Aug 12 2009, 08:35 AM, said:

There are many threads on contamination in the cell biology sub-forum.  Basically it boils down to use the immunofluorescence method and it is best to send them away to an experienced company (which is quite expensive, but worth it).

Thank you very much!! I will try to check the contamination!!

Thanks!!!

[flysciguy]

Hi, I'm also trying to expand the same CD40L-3T3 cells for culturing B cells. Did you ever have any success with these cells? My real problem is that after adding PBMCs to the 3T3s, the 3T3s will come off the plate. But the 3T3s without PBMCs stay attached.. Do you have any tips?

Thanks for your help,
-Alex