Hi Joshua,
That is very helpful. Can I ask what vitality parameter(s) you used?
Mike
Hi once again Joshua
Sorry to drill down further down here, but what concentrations of the lyso dye did you try and were you continuously imaging for 5 mins or merely staining? The later is a concern as most dyes will become toxic if exposed to light for very long. I really appreciate the details.
Mike
I have followed the Invitrogen protocol and incubated 10nM and 20 nM concentration of lysotracker (Red DND-99) adherent cells at 37oC in a serum free medium without phenol red for
I am interested in studying the phagosome-lysosome fusion as well. We have some lysotracker (Red DND-99). The manual says incubation time upto 5 min.
Others are talking about hours of incubation.
I wonder what would be an ideal time of incubation. i will have cells on a glass slide. My questions revolve around 1) time of incubation 2) fixing or not fixing and 3) washing steps. Any comments would be appreciated. MK
To Mayur,
I have been using lysotracker red on my primary neurons to compare the intracellular pH between cells transfected with different constructs. The incubation time is 30 mins with fixing. According to the manufacturer, incubation longer than 5 mins would cause alkalizing effect. For this I cannot comment because I haven't done a different incubation period in parallel to compare it to my 30 mins incubation set. Nevertheless, we still see the predicted significant change of pH between treatment group and rescue group with 30 mins incubation. It seems that even if the prolonged incubation might have effects in intracellular pH, the effect wouldn't interfere much with the end result if control and treatment group are done in parallel.
so here is my response:
1) 30mins
2) it can either be fixed or not fixed. the important point is to avoid detergent at all cost. it would quench almost all LTR signal within seconds. (for this point, actually i am still open for opinion or evidence from others. anyway this would be my recommendation until i see evidence that points to the otherwise)
3) i wash it 2x with warm culture media because primary neurons are highly susceptible to change of cultural environment such as osmolarity and all that and will shrink (die) immediate upon any shock. I think PBS is suitable for subculture line or other cell types that is more robust.
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