I have administered CQ treatment (50 mg/kg) in twelve-months-old C57BL/6J mice to assess autophagic flux. I validated this protocol on 3-months-old mice first, obtaining an accumulation of LC3II by WB, which I don't see in 12-months-old mice. Actually, there was a decrease in LC3II levels after treatment.
How could it be possibile?
Hi,
Theoretically you should indeed have inhibition with this dose in the 10-100 mg/kg window although 100 mg/kg i.p. seems to be the recommended dose these days.
How long after treatment did you collect the tissues? Did you perform the 2 experiments at the same time of the day? Autophagy has an important diurnal rhythm and you may not see the same LC3-II accumulation upon lysosmal degradation blockade early in the morning than in the afternoon (See Fig 1B). Article Temporal orchestration of circadian autophagy rhythm by C/EBP??
Or more straight forward and technical, your 12-month mice are bigger and fatter are you sure the injection did indeed reach the peritoneum? I was taught i.p. is not the most reliable and injection in abdominal fat, gut or even subcutanous can happen.
Dear Dr. Albanese,
Dr. Pouly provided a good answer,
The only thing that I could add is that
older mice tend to have "an aged" immune responses
vs 3-6 month old mouse whom have a robust immune system.
Best Wishes!
Hi,
There can be few issues. Tissue processing affects these proteins. More importantly, in these mice there may be altogether a reduced flux through autophagy pathway (as compared to 3 month old mice).