Autophagy assay cell type - A2780

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 6 matching solutions for this experiment

Downstream tips
- Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time.

- Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.
SQSTM1/p62 (D5E2) Rabbit mAb

Cell Signaling Technology

Upstream tips
- Lyse cells in RIPA buffer containing 10% phosphatase inhibitor and 10% protease inhibitor cocktail.
Protocol tips
- Dilute primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

- Dilute primary Ab at 1:1000
SQSTM1/p62 Antibody

Cell Signaling Technology

Protocol tips
- Dilute primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

- Dilute primary Ab at 1:1000
LC3A/B Antibody

Cell Signaling Technology

Protocol tips
- Dilute primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

- Dilute primary Ab at 1:1000
Atg14 Antibody

Cell Signaling Technology

Protocol tips
- Dilute primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

- Dilute primary Ab at 1:1000
Beclin 1 Antibody

Proteintech Group

Protocol tips
- Dilute Ab at 1:10000 in blocking solution.

- incubate primary antibodies overnight at 4°C.
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