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HTC cells not attaching to flasks

macrophage attaching HTC

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#1 matridium

matridium

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Posted 31 May 2012 - 06:16 PM

I have been given the task of growing some macrophages for a corrlative study by our group. I got a T25 of immortalized chicken HTC cells from a fellow lab. The first passage of the cells went well and the cells were happy, and attached and growing.
The second passage went well too, and I freezed down several flasks for LTS. Third passage the cells did not grow well at all, they balled up, did not attach well, however they did grow. what I'm perplexed about is the fact that they attached at first and now they do not.
On top of this every vial, (3) that I have thawed out exhibits the same behavior. And I asked for a new flask of cells and I was given a T75 flask I split it 1:10 in T25 flasks over a weekend. I came back and I had plenty of growth but no attachment.

dissociation: Tryple express
Freezing media 10%DMSO and 90%MINIMAL I was told later to use 10%DMSO/90% FBS

We need the cells to attache to cover glass so we can feed beads to them.
I am thinking either I have used the tryple for too long, or the freezing media is to blaim, however the second batch after dissociation did not reattache after passaging. so maybe the tryple is to blaim.. I'm actually at a loss.

any ideads?

Mike





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