Ask: I want to share the results of my experiment to confirm the transfection rate of hek 293 cells (suspended not adherent) with a plasmid that reports with GFP, 2 days and 5 days after transfection, I have 2 problems:1- cells changing their scattering from day to day (I am not sure if my gating was right or not). 2- The cells gave high fluorescence 4 days after transfection, it worth to say, when I directly ran flow without processing of cells (without wash, pbs or even spindown), the autofluorescence disappeared, is it something related to the dead cells.
Hi Haitham,
How do they look in the microscope? Both in the DIC and GFP ... maybe the amount of DNA you have transfected is too much and being toxic. This toxicity could be increasing both your SSC and consequently your autofluorescence in the green channel. Check out this discussion in researchgate, it may be helpful for your assay:
https://www.researchgate.net/post/Do_you_have_experience_in_HEK293_transfection_in_6_well_plates
All the best,
Nicolle
Hi Haitham, I will take advantage of Ceri's answer to respond both of your questions. I agree it is very important to have a look on your non-transfected control first of all. How does your normal 293 culture look like in the FSC and SSC? You should use it to clear all your doubts about log/linear and where they should be placed. In the linear graphs you showed me ( in the other question posted) I think you have low counts because there are a lot of events outside your view, possibly cell debris. Review if the method you're using to harvest them from the culture/wash/centrifuge is not too harsh.
Another important question I would like to ask you is: are you sure your control vector does not contain GFP? If so, why not? Usually when we are transfecting cells we use an "empty" vector but which also expresses GFP alone, since GFP expression by itself can alter cell processes. It is also a better positive control - a good way of knowing if your transfection is working ok, since a vector with only GFP is smaller and is more efficiently inserted in cells than larger vectors with GFP+gene of interest.
Good luck!!!
Thank you very much dear Ceri, I tried actually to gate for the living cells but the autofluorescence even of the non-transfected cells made the histogram messy, I am using PEI as a transfection reagent, Is there any difference between FITC-A and H? I don't know this piece of information.
Thanks Nicolle for your concern, Normal untransfected hek 293 cells (suspended not adherent) scatter was changed from days to day, Yes I have low count and so I was worried about using the linear graph and If I have some cells that I consider it as debris, for harvesting, I use pbs, spin it down (500g, 5 min. 4 C).
Dear Haitham,
to set up a flow cytometer the first time for a new cell type can be tricky (what flow cytometer are you using?). I would also recommend using either propidium iodide or DAPI/Hoechst to differentiate live/dead cells (depends on the lasers your flow cytometer has). PI needs some compensation with GFP though. Also, I'd reduce the g force in your spins to 300 g.
FSC and SSC are best aquired in linear mode. I can't really tell much from your figures but they suggest your set-up is not right, perhaps you also have lot's of cells clumps...
Can you ask an experienced FACS user to help you with the set-up?
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