Hello,

I am a new phD student and I am trying to assess a protocol for evaluating autophagic flux in mouse spleen (or others), even if my real experiment animal model will be rats.

I was wondering if someone had some experiences with Guava Autophagy kit and, in case, how you performed the staining starting from spleen homogenate.

Basically I have two major question arising, would you resuspend the spleen homogenate in PBS or in growth medium (i.e. RPMI+ 10% FBS)?

Then, if I want to test CQ effect on autophagic flux but the kit also suggests the incubation of autophagic reagent A to be able to read LC3B signal, how should I proceed? Should I perform before the incubation of this reagent and thereafter CQ incubation or the opposite?

I would thank you in advance for any tips or recommendations.