I have a gene knocked down by siRNA technique in my stromal cells. After confirming the down regulation of the gene I have done a serial dilution of the cells to seed them on a 96 well plate for single cell cloning. After reaching confluency in some wells, these cells were subjected to flow cytometric analysis.
I have observed that I have to gate it differently compared to that of the parental cells and also a new population appeared in the picture though there is no procedure I have mistakenly done for contamination during the process of cell seeding, maintenance or flow cytometry. I have taken the cells from one of the wells from the 96 well plate and grown on a 6 well plate for maximising the area of growth. They still have the changed morphology rather than the original/parental cells which are used for the knock down. I am interested in knowing the reason for this.
Hi Prabhu,
since you didn't tell which context your knockdown target is relevant for, it is difficult to give useful advice for your question.
The best suggestion I can give would be to do the same procedure as you described above for the parental cell line without knockdown (In case you havn't done yet). I guess your are not interested in investigating to study the effect of passage number etc.
Best,
Kai
Also curious about what siRNA controls were performed. Delivery lipids alone etc... More info could help us help you.
Cheers :-)
Hi Prabhu,
Do you have the same effect in other cell line or it is specific for your stromal cells? or have you tried using other siRNA duplex targeting other unrelated gene? Probably the protein product of your target is involved in cell morphology... Good luck
Cheers,
Ricardo
Hi, Ka,i Steven and Ricardo are all correct and should be added to your experiment, especially the scramble probe for a transfection control. If you incorporate the parental line, with a sramble control and your siRNA set, for 2 cell lines you should have your answer. If it happens to all cells then it might be the culture conditions. If the changes occur with both your control and siRNA transfections, then it's a product of transfection. If it only occurs in the siRNA samples then your protein targets may be involved with cell morphology.If it occurs only in one cell line but not the other, then that one cell line may be more sensitive to the previously mentioned conditions. Have fun!
Hello all, Thank you all for your answers. Since, I have not done all those which you have mentioned above, I will perform them and sort it and if still the problem persists, I will contact here again. Thanks again.
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