Autophagy assay cell type - HepG2

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 15 matching solutions for this experiment

Upstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
Resuspend 5 × 10*5 cells in 1× assay buffer and incubate with the Cyto‐ID dye at 37 ºC for 30 minutes in the dark, followed by flow‐cytometric analysis. For Fluorescence microscopy, grow the cells on coverslips after respective treatment incubate them with the dual detection reagent (Cyto‐ID dye and Hoechst 3342 nuclear stain) at 37 ºC for 30 minutes in the dark.
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.
Upstream tips
Lyse cells in ice-cold mammalian lysis buffer
Protocol tips
For WB, incubate with primary antibody diluted to 2 ug/ml in total volume of 3 mL in Blocking
buffer for one hour at room temperature.

For IF, stain with primary antibody for 45 min at RT in 40 ml of 1%BSA-PBS by forming a
drop on the coverslip
Beclin-1 (D40C5) Rabbit mAb

Cell Signaling Technology

Protocol tips
For WB, dilute primary Ab at 1:1000 and for immunoprecipitation at 1:100.

For western blots, incubate membrane with diluted antibody in 5% w/v BSA, 1X TBS,
0.1% Tween®20 at 4°C with gentle shaking, overnight
SQSTM1 Antibody (D-3)

Santa Cruz Biotechnology

Protocol tips
For Western Blotting (starting dilution 1:100, dilution range 1:100-1:1000.

For Immunofluorescent staining incubate with primary antibody at 8°C overnight.
Protocol tips
Dilute primary antibody at 1:1,000 dilution and incubate overnight at 4°C
Protocol tips
Dilute primary Ab between 1:500 - 1:1000 and incubate overnight at 4°C
Protocol tips
Dilute primary Ab between 1:500 - 1:1000 and incubate overnight at 4°C
Protocol tips
Dilute primary Ab at 1:2000 and incubate at overnight at 4°C.
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C
LC3B (D11) XP® Rabbit mAb

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C
PI3 Kinase p85 Antibody

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C
Protocol tips
For WB, dilute primary Ab between 1:10000 - 1:50000
Protocol tips
Use a concentration of 1 - 5 µg/ml.
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