Autophagy assay cell type - K562 cells (human myelogenous Leukemia)

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.

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 9 matching solutions for this experiment

Protocol tips
The autophagy-related LC3 protein (ENZO) in cells
was detected using Cyto-ID. Cells were grown on the
coverslip and selected when the density was 50-70%,
followed by intervention according to the experimental design; the negative control group was set up. The
supernatant was discarded and the cells were washed
twice with 1×Assay buffer for 30 min and were washed
with 1×Assay buffer, and the excess buffer was removed,
followed by sealing using anti-quenching agent containing 4’6-diamidino-2-phenylindole (DAPI) dye. Then, cells were incubated in the dark at 37ºC . The coverslip was observed under fluorescence microscope (FITC and DAPI; 60×).
Protocol tips
For Western blot (WB) and immnufluorescence, we employed: rabbit anti-LRP1β (Sigma–Aldrich Canada), rabbit anti-LC3B (Sigma–Aldrich), mouse anti-β-actin (GenScript), mouse anti-GM130 (Sigma), anti-rabbit HRP (Sigma), mouse anti-HRP (Sigma), Alexa Flour 488 anti-rabbit (Sigma), Cy3 anti-rabbit (Sigma), Cy3 anti-mouse, and Alexa Flour 594 anti-rabbit (Sigma).
Protocol tips
Cells were analyzed for autophagy induction 48 h after transfection using FlowCellect™ Autophagy LC3 Antibody-based Assay Kit (Merck Millipore). For autophagy induction, the cells were either starved for 3 h (induced) or left untreated (control).
Atg5 (D5F5U) Rabbit mAb

Cell Signaling Technology

Upstream tips
Lyse cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride and 1 mM NaF) containing complete protease inhibitor cocktail
Protocol tips
Dilute primary antibody at 1:1,000 dilutionand incubate at 25°C for 2 h
Beclin-1 (D40C5) Rabbit mAb

Cell Signaling Technology

Upstream tips
Lyse cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride and 1 mM NaF) containing complete protease inhibitor cocktail
Protocol tips
Dilute primary antibody at 1:1,000 dilutionand incubate at 25°C for 2 h
LC3B Antibody Kit for Autophagy

Thermo Fisher Scientific

Protocol tips
Dilute the LC3B rabbit polyclonal antibody in blocking buffer to prepare 0.5 μg/mL working solution.
LC3A/B (D3U4C) XP® Rabbit mAb

Cell Signaling Technology

Upstream tips
Lyse cells in RIPA buffer containing 1 mM PMSF
Protocol tips
Dilute primary antibody at 1:400, 4 °C overnight.
SQSTM1/p62 Antibody

Cell Signaling Technology

Upstream tips
Lyse cells in RIPA buffer containing 1 mM PMSF
Protocol tips
Dilute primary antibody at 1:400, 4 °C overnight.
SQSTM1 Antibody (D-3)

Santa Cruz Biotechnology

Upstream tips
For optimal results, use low fluorescence PVDF membranes
Protocol tips
Dilute Ab at 1:200
Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!
Become shareholder Discussions About us Contact Privacy Terms