I used a GFP vector to evaluate the transfection efficiency. the negative control, is a GFP-free vector. results of Fluorescent microscopy show transfection at rates higher than 90%. But the results of flow cytometry show transfection rate is about 40%.
What is the reason for this mismatch?
The advantage of FCM is the large number of cells scored. The problem, as noted above, is that different gating or different groups can give you aberant results. If you used the same gates of FS (cell size) and SS (granularity), for unexposed, GFP transfected and vector only transfected cells, you should be seeing the same cells in each population; then the fraction of GFP positive cells in the GFP vector transfected cells should be an acurate measure of your transfection efficiency (assuming you gate GFP to capture all GFP positive cells, i.e., your background fluorescence should be set by that of the other two groups). FM is subject to the problems of uneven staining over the areas measured, relatively low number of cells scored, etc. Unless some staining intensity (automated image analysis) measurements are made, the results tend to be more subjective; e.g., the difference between Pathologists scoring the same slides.
Did you gate your cells in the SSC and FSC? For flow, you cannot take all events, if you have gate your cells.
Otherwise, you might be setting your positive and negative gates incorrectly.
These are the only things I can think of.
Hi Marzieh, I have a big problem with flowCy. You can get any result you want by selective gating.
Trust the fluorescent microscopy data more than flowCy..
Estimates of transfection efficiency may be misleading even under fluorescence microscopy, due to image quality, filters, etc. Do you really need an ABSOLUTE value of transfection efficiency? Otherwise, you can design your experiments based on comparing results of GFP-transduced vs. empty vector-control cells.
Rafael Linden that's what Marzieh has done. Read her question.
Hi ,
Cell Sorting or gating the transfected cells normally gives the accurate value by FACS analysis. Lot of false background is expected in case of normal fluorescent microscopy compared to flow cytometry which is evident in your results of more than 90% compared to the results of flow cytometry show transfection rate is about 40%.
Good luck !
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