Harvesting cells - (Mar/13/2014 )
Can excess trypsinization kill cells during harvesting?
Yep, it can. You can check the cells under the microscope however to see when the majority are detached and when you need to stop the trypsin reaction by adding medium.
I added Trypsin and I think it's too much. I had added 1 ml of trypsin for cells plated on a 6 well plate and didn't add medium to stop the reaction. But tried to wash the cells with PBS and I found a clump formation after the centrifugation. I checked the cells with viability dye and found all were dead after flowcytometry analysis. I prepared these cells after a transfection experiment.
Thank you very much
This could be your problem then, you could try other trypsinization conditions to find out. It's not just about how much trypsin is applied, but also about how long. How long did you live the trypsin on, and was it at 37 °C ?
As previously stated over trypsinisation will cause damage/death of the cells. The variables are:
Concentration of the Trypsin
Length of exposure
Most protocols indicate that the trypsin should be in contact with the cells for as short a time as possible. It should act as efficiently as possible which means that we warm up the trypsin solution to 37oC. We also wash the cell monolayer with PBS without Calcium and Magnesium. Both these divalent cations inhibit trypsin activity
You can also use less aggressive enzymes to sub culture cells i.e. Pronase, Elastase, Hyaluroniadase, collagenase etc
I had incubated the cells at 37 degrees for 5 minutes.
Thank you very much all of you, I started repeating the experiment today, by Thursday I will be able to give u all some feedback. and I will be taking these points stated here.
I repeated the transfection for my epithelial cell lines ( two cancer cell lines and a control normal epithelial cells line), This time cells were all alive. The problem was with the trpsinization reaction as last time I didn't stop using serum. I id the transfection with pMAX-GFP and turbofectin 8. Unfortunately there was a very low level of transfection (27 % fluorescence) in the normal control epithelial cell line and nothing in the cancer epithelial cell lines after FACS analysis. pMAX-GFP available at the lab is quite old, 2 years,Could not figure out the problem still. But in the protocol it is mentioned to use Opti-Mem reduced medium, but I had to use serum free media at the time of my experiment.
I have repeated the the transfection with cancer cells of epithelial origin (MCF -7, MDA-MB-231, BT-20) using Turbofectin 8 and with pRFP-C-RS and pmax-GFP but ended with a very low transfection efficiency. My cells were live but with no transfection.
Hope you all could help me
So - what did you do? - we need details for any sort of troubleshooting.