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Cell detachment in clonogenic assay

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#1 JDSBlueDevl



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Posted 13 May 2016 - 02:32 PM

I'm doing a clonogenic assay in which I transfect cells with my plasmid of choice, then wait two days to do irradiation with single-cell suspensions at 0-6 Gy and replate afterwards.  I made sure to count my 184A1 cells before I irradiated and apportioned accordingly (I used a density of 3.6*10^3 in 1 mL medium in a conical tube).  After irradiation, I made sure to replate in a 12-well dish with counts of 0, 100, 500, 1000, and 2000 cells in a well (yes, this took more than one plate).  After two days, it looked like I was getting some good attachment and even some initial cell division in some wells (184A1 cells have a long doubling time), so I decided to change the medium.  Afterwards, I saw that I lost my cells to either the aspirator pipette or detachment with a 10-mL pipette when dispensing fresh medium.  I'm trying to take account of the mistakes I made here and what I could do next time.  I know that when I aspirated the post-transfection plate prior to trypsinization, I did not have any issues with detachment.  Of course, I forgot whether I used a P1000 tip on the aspirator pipette or not.  Another is if I should be using P1000 micropipette tips to dispense my medium instead of a 10-mL pipette.  Of course, there is also the question of how much time my cells were in suspension (3x5 min trypsin treatments, neutralize 1:1 with PBS containing 5% FBS, spin down 5 min at 500 g, resuspend in medium, where it sits for ~40 min while I count, apportion, irradiate, and re-plate).  I've been told that as long as the trypsin has been neutralized and/or removed, cells being in suspension for 1 hr shouldn't be a problem.  Someone else suggested coating my wells with collagen I, but I'm leery of whether this could affect my results.  Can someone help me make sense of this?

Edited by JDSBlueDevl, 13 May 2016 - 02:48 PM.

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