Autophagy assay cell type - HL-1

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 3 matching solutions for this experiment

3-Methyladenine (3-MA)

Selleckchem.com

Protocol tips
After treatments, MAEC and HL-1 cells were washed three times with cold PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing the Protease Inhibitor Cocktail and protease inhibitor PMSF for 30 min. Lysates were centrifuged at 12000rpm for 5 min at 4 °C. The protein samples were boiled in SDS-loading buffer for 10 min and were separated by 10% SDS-PAGE. Immunoblotting was performed with primary antibodies against parkin (1: 500), LC3 (1: 1000), P62 (1: 1000), ERRα (1: 1000), eNOS (1: 200), p-eNOS (1: 200), TOM20 (1: 1000), cleaved-caspase-3 (1: 1000), and GAPDH (1: 1000) followed by the incubation with HRP-labeled secondary antibody. Protein bands were visualized using ECL Plus detection reagents with Amersham ECL Detection Systems (Amersham, Buckinghamshire, UK). Densitometric analysis was performed using Image Lab Software (Bio-Rad, USA).
Bafilomycin A1(Baf-A1)

Selleckchem.com

Protocol tips
After treatments, MAEC and HL-1 cells were washed three times with cold PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing the Protease Inhibitor Cocktail and protease inhibitor PMSF for 30 min. Lysates were centrifuged at 12000rpm for 5 min at 4 °C. The protein samples were boiled in SDS-loading buffer for 10 min and were separated by 10% SDS-PAGE. Immunoblotting was performed with primary antibodies against parkin (1: 500), LC3 (1: 1000), P62 (1: 1000), ERRα (1: 1000), eNOS (1: 200), p-eNOS (1: 200), TOM20 (1: 1000), cleaved-caspase-3 (1: 1000), and GAPDH (1: 1000) followed by the incubation with HRP-labeled secondary antibody. Protein bands were visualized using ECL Plus detection reagents with Amersham ECL Detection Systems (Amersham, Buckinghamshire, UK). Densitometric analysis was performed using Image Lab Software (Bio-Rad, USA).
Upstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min
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.
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