Primary lung fibroblasts not lifting off

[steven_wong_05]

Hi everyone,

I culture Balb/c and C57Bl/6 mouse lung fibroblasts in MEMF15 media containing 10% FBS. I am having a lot of difficulty lifting off the cells when I want to passage them. I use trypsin, but recently have moved to 1x Tryple Express, which is preheated prior to use at 37*C. After 20 minutes (quite long!), I notice that at most 40% of my cells have lifted off. This situation is quite peculiar since I have worked with a different batch of Balb/c and C57Bl/6 mouse lung fibroblast in the past and they lifted off more efficiently than these batches of cells.

Here is the protocol that I use to trypsinize my cells:
1) Remove media from flask
2) Wash flask with 1xPBS to remove residual FBS
3) Add 1 mL of preheated trypsin or Tryple Express to cells
4) Place into 37*C incubator

I have tried a new stock of Trypsin, changed my 1xPBS stock solution, so do not know what to do. Does anyone have any recommendations?

[relaxin]

If you use T25 flasks, you may need 2 ml of TrypLE instead of 1 ml. TrypLE works better than trypsin, because it is collagenase-based. You may rinse the cells with 2 ml of TrypLE, and then add 2 ml of fresh TrypLE and incubate until all cells are dettached. Agitation may be needed.

[CrowSan]

and make sure that your using the right PBS! Although a quick wash with trypsin prior to trypsinisation (as Relaxin suggested) will get rid of this in case it is the wrong type (with Mg ca)