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I'm interested to analyse hemolymph cells from a marine mollusc. I wanna count only "alive" cells so my strategy was the following: DRAQ5+ (nucleated cells) and PI- (alive cells) and identify population in FSC vs SSC.

Staining independently with DRAQ5 and PI I found positive and negative populations for each one but, in the fully staining I lost DRAQ5+/PI+ population... (https://ibb.co/VpgcCTN)

Does anyone know if DRAQ5 and PI are incompatible?

Note:

  • DRAQ5: excitation with 640nm and emission filter 675/25.

  • PI: excitation with 488nm and emission filter 585/40

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  • $\begingroup$ Rieger et al. 2010 seems to be relevant. -- "Gating on the population of PI+/DRAQ5+ cells, we used the similarity feature to determine the degree of similarity between PI and DRAQ5 stains in goldfish primary kidney macrophages (Fig. 3A). We found that there was a sizable proportion of falsely stained PI positive in these cells, as well as in all unfixed primary cells and cell lines tested (Fig. 3B)." $\endgroup$
    – acvill
    Jan 27, 2020 at 14:18
  • $\begingroup$ For my proposal, I prefer to have more false-positive than false-negative cases. The problem here is that I don't have double staining as you can see here. Independently, I have DRAQ5+ and DRAQ5- populations, the same happens with PI, I saw PI+ and PI- populations. But, adding both stains together I lost DRAQ5+/PI+ population... $\endgroup$ Jan 28, 2020 at 18:43

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