Dear all,
I am trying to determine the DNA content in cell population by Hoechst 33342 DNA specific stain using flow cytometry analysis (BD FACS fortessa). I am observing two different clouds ( marked in red and blue in below picture) in the DNA content vs Side scatter dot plot. Does anyone know what are these two distinct clouds represent? Does top and below (blue and red respectively) represents dividing and non dividing cells? Any suggestions, explanation and literature reference will be highly helpful. Anticipating an early response!
Best regards,
Ankur
Hi Ankur,
DNA content is run on a linear scale rather than log scale. If you switch that DAPI channel to linear on your next run it should give you a normal rendering.
Best,
Rich
Hi Richard, thanks for your suggestion. I have already tried that during my experiment. I can show you FlowJo based analysis which shows no significant difference when I switch from log to linear and vice versa. I don't know if it is very common or I am seeing it due to heterogeneity in "tissue" sample.
Oh, I see. Is it possible that your cells are re-aggregating during the prep or run and creating random size clusters? That would give you a series of progressive multimers making a population every so often down the axis, with each cells additional DNA. I'm sure someone with more expertise in DNA content analysis can give you a better answer, but it's fun to guess :)
I think are getting clumps in your sample. You need to gate single cells for proper data. How much careful you are during sample preparation you would end you have double cells. The reason being dead cells have sticky DNA which are responsible for this problem. So after your SSC-A and FSC-A you need to doublet discriminate and only gate the single cells. For doing that have a graph as FSC-H (PI-H) and FSC-A (PI-A), then select the upper portion of the cell mass. i think it should help you with your problem and eliminate your doublet cells and cells that look odd.have a look at this image.
https://www.google.co.in/search?q=doublet+discrimination&source=lnms&tbm=isch&sa=X&ved=0ahUKEwiQkeem84nbAhUNO7wKHYDWBiAQ_AUICigB&biw=1366&bih=637#imgrc=PuBmeIx3ixpe9M:
- Badges
- Science method