I am processing and disgregating patient-derived tumor samples for flow cytometry analysis. I use anti-Human CD45 positive to characterize the immune cells within the tumor but not sure what is the best / most recommended way to report immune infiltration percentage: CD45+ of live? CD45+live of total cells? CD45+ of total cells?
Hello
The most suitable way to report the percentage of immune cells is from a CD45 vs SSC gate, because the reference values come from that plot in almost all previous researches. So you must first positively select the live cells in a plot of viability vs SSC, then from the live cells create a CD45 vs SSC plot. Then, from live cells and CD45+, determine all the percentages of immune cells. If you wish, you can consult the article "Estimating Normal Values of Rare T-Lymphocyte Populations in Peripheral Blood of Healthy Cuban Adults" on my profile, in figure 1 you can see the gating strategy, in that case the viability plot is missing but you can insert it as I said between those of FCS vs SSC and CD45 vs SSC.
Carlos Agustín Villegas-Valverde
Your answer was useful for me, but could you please let me know what’s the most recommended way to count and represent the cell number ? thank you in advance
Hello Rayene Benlaribi,
If you refer to the absolute cell count, the most suitable way to count cells in cytometry is using the single platform with counting beads. The single platform has several advantages over the double platform, that is the largest international consensus.
Each manufacturer has their fluorescent microspheres or stabilized cells for absolute count, I am working with the Flow-Count ™ Fluorospheres from Beckman Coulter. its use is very easy.
Usually in your acquisition protocol you create a gate that names "CAL" in a dot-plot of SSC vs FL (fluorescence channel where the beads emit) or you can also use Time vs FL, this is the one that I usually use since it has the advantage visualize the acquisition stability of the counting beads. Finally, in your acquisition and / or analysis software, you should have the option of introducing the beads concentration factor, which is provided by the manufacturer and in the statistics menu, enable the absolute number option and thus obtain the absolute cell count.
the calculation formula is:
Absolute Count (cells/μL) = Total Number of Cells Counted / Total Number of Fluorospheres Counted x Flow-CountFluorospheres Assayed Concentration