Autophagy assay cell type - CaCo-2

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 8 matching solutions for this experiment

Upstream tips
Chloroquine, a well-known reagent able to block autophagic flux and cause an accumulation of autophagosome in the cytoplasm, is used to treat Caco2 cells to use as positive control.
Beclin-1 (D40C5) Rabbit mAb

Cell Signaling Technology

Protocol tips
- dilute primary antibody at 1:1000 in 5% w/v BSA, 1X TBS, 0.1% Tween 20 at 4°C with gentle shaking, overnight.
LC3A/B (D3U4C) XP® Rabbit mAb #12741

Cell Signaling Technology

Protocol tips
- dilute primary antibody at 1:1000 in 5% w/v BSA, 1X TBS, 0.1% Tween 20 at 4°C with gentle shaking, overnight.
Protocol tips
- For IHC, use promary Ab dilution at 1:600 and for Western Blot use between 1:1,000 – 1:3,000
SQSTM1 Antibody (D-3)

Santa Cruz Biotechnology

Upstream tips
- Lyse cells in M-PER Mammalian Protein Extraction Reagent
Protocol tips
- Use a dilution range 1:1001:1000
Upstream tips
- Lyse cells in M-PER Mammalian Protein Extraction Reagent
Protocol tips
- Use a dilution range 1:1001:1000
Upstream tips
- Lyse cells in M-PER Mammalian Protein Extraction Reagent
Protocol tips
- Use a dilution range 1:1001:1000
Protocol tips
- Use primary Ab dilution at 1:200
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