Autophagy assay cell type - RAW 264.7

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

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Found 7 matching solutions for this experiment

LC3B Antibody

Novus Biologicals

Protocol tips
Cells were cultured in normal culture medium under normoxia (21% O2) or hypoxia (1% O2) conditions for the indicated times. Cells were subsequently lysed in RIPA lysis buffer containing protease inhibitors and subjected to immunoblotting with primary antibodies
Protocol tips
Dilute primary antibody in 1% normal serum or BSA (in 1X PBS). Incubate coverslips with ~5 µ g/mL rabbit anti-LC3 primary antibody (NB100-2220) for 1 hour at room temperature (37°C is optional), or for 16 hours at 4°C.
Protocol tips
Incubate cells with both primary antibodies in 1% BSA in PBST in a humidified chamber for 1 h at room temperature or overnight at 4°C.
ATG12 Antibody (166)

Novus Biologicals

Protocol tips
For WB dilute primary Ab between Western Blot 1:500-1:3000 and incubate overnight at 4 °C
Protocol tips
Dilute primary Ab between 1:300-1000 and incubate overnight at 4 °C
LC3A/B (D3U4C) XP® Rabbit mAb

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C
Protocol tips
Incubate primary Ab overnight at 4 °C
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