I analyzed the data of my flow cytometry assay.
Data of Capture-1 is about cell apoptosis induced by nanoparticles in cell culture.
1. Was the gate set correctly?
2. Why did the clusters of the live cells, apoptotic cells and necrotic cells stick together?
3. Should I use the percentage of Q4 to make bars representing cells apoptosis or Q2+Q4, I really need suggestion.
Data of Capture-2 is about the nanoparticle delivery RNA labeled fluorescence.
1. Was the gate set correctly?
2. Why P1 and p2 have same number?
3. Where did number 1,341 (down right corner) come from?
4. Should I use p2 or Median 1,341 to represent labeled cells?
Thanks
Attached are two files of capyure-1 and capture-2
Capture 1:
1. Looks okay but you might be excluding dying/dead cells at the bottom left corner. Important since you are trying to look at cell death.
2. There won’t be a clear separation between all your cells.
3. Cannot say until we know what your axes represent (FITC and PE).
Capture 2:
1. Again, cannot say as we don’t know what your staining was in your untreated (non-dead) sample was.
2. Basically, all your cells in P1 are also in P2 and I am not sure you are gating P2 properly. It could be positive or negative for P2 (again need untreated cells to say one way or other). Also, what is on PE-Texas Red?
3. It is the media value (Fluorescence intensity) for the P2 histogram
4. Depends on what you want to show
All in all, a few suggestions.
1. Add proper controls if you didn’t have them already (like the others suggested). This would include unstained cells, cells stained with single colors and FMO controls. Also have stained cells where you don’t expect to see cell death (unstimulated/non-treated) so you know how to gate for cell death markers.
2. I would suggest that you find someone in your lab/dept who is an expert in Flow Cytometry and get them to explain the technique and proper gating strategies to you. It is important that you know how to analyze flow data properly so you don’t make interpretations that are logically and scientifically problematic.
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