I haven't run into this issue before, but you should find out what lazars were in use, mcherry excites with different wave lengths, and I am assuming the resultant emission spectra are different as well and one might bleed into the dapi channel

Dear Alex!

You look at following article, please:

Article Soting cells for basal and induced autophagic flux by quanti...

Figure 2. Quantification of autophagic flux by flow cytometry. (A) Example of flow-cytometry data illustrating flow cytometer setup for measuring autophagic flux. Scatter and singlet gates should be used to eliminate debris, dead cells, and mitotic cells. Voltages and gain on GFP and mCherry detectors are set empirically to allow both negative and positive control (starvation) plots to fit the mCherry/GFP ratio histogram. (B) Comparison of this protocol to other previously published methods for flow cytometric quantification of autophagy. C-G-LC3 reporter cells were starved for 4 h in EBSS followed by flow cytometry with or without extraction of cytosolic (nonlipidated) LC3 by saponin. (C) Concurrent measurement of autophagic flux and apoptosis using the established flow cytometry markers ANXA5/annexinV and DAPI. C-G-LC3 reporter cells were treated with an apoptotic stimulus (Fas ligand) for 4 h and stained with ANXA5/annexinV-APC and DAPI followed by flow cytometry.

3.3.1 Flow cytometer setup

Proper setup of the flow cytometer is a critical step of this protocol. Each cytometer and associated acquisition software have their own quirks, especially with regard to handling of ratiometric parameters. It may prove valuable to get help from someone with experience using ratiometric parameters on the machine being used. Here, we will describe setup on the XDP-100 sorting flow cytometer (Fig. 2A).

Forward scatter/side scatter and doublet discrimination should be set according to the parameters most applicable to the flow cytometer being used, and detector voltages set for the particular cells being used. As depicted in Figure 2A, following FSC-Height and SSC-Height, SSC-Width is used to gate singlets on the XDP-100 sorting flow cytometer. These gates result in the mCherry-GFP dot plots and ratiometric histograms shown for control and EBSS treated cells, respectively.

We have found that on the XDP sorters, using linear input parameters rather than log is most useful for the derived ratiometric mCherry/GFP parameter. However, this may not work best for all machines and is not possible with some. It is sometimes difficult to keep both the negative and positive control conditions on scale for the ratiometric parameter, unless the software is capable of using derived, higher-level mathematical parameters. For most applications, the user must set the voltages/gain for both the mCherry and GFP parameters carefully to get the ratio parameter on scale and with good dynamic range. Generally, this means getting the GFP channel just below the middle of its range with the mCherry channel just above the middle of its range (Fig. 2A). This results in a low positive number that can be tweaked from experiment to experiment to optimize the scale and dynamic range. It should be noted that using voltages too low will decrease the dynamic range, but voltages that are too high increase the noise. Getting the balance just right sometimes takes some trial and error and varies greatly from cytometer to cytometer. We have found that this is easiest to do by first adjusting voltage using a positive control for flux (like EBSS starvation) to get those on scale, then using negative control (either basal or flux-inhibited) cells and adjusting to get those on scale. Once the voltage and gain are set on a particular machine they should be noted and used as a starting point for the next experiment, though they will frequently need to be adjusted.