I am new to Lysotracker staining and I'm not quate sure what do I do wrong so I'm kindly asking if anyone can help me.

I'm working with primary HDF of healthy donors and mucopolysacharidosis patients.

I choose concrentration of 100 nM of Lysotracker and incubation time of one hour.

Then I wash 2 times, fis with 4% PFA for 15 min, then wash 3 times (with PBS), then stain with Hoechst for 10 min, then swash again 3 times, and the mount.

then analyse it with just fluorescent microscopy.

I've had several problems:

1) I use 8-welll chamberslide and sometimes after staining and fixation I don't have any cells in some of the chambers

2) around the cells there are tiny dots, what are these? are they metabolites or mycoplasma? same was observed with lysosensor but not with nile red, so i'm confused a bit, not even sure what exactly stains them - the lysotracker/lysosensor or hoechst?

3) on the picture there is cells from a healthy donor, and it clearly has 2 populations - one with enhanced lysotracker staining and the other one is not so much. why can it happen?

I would appreciate if someone would help me with protocol and explain the picture!