Autophagy assay cell type - A549

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 12 matching solutions for this experiment

Anti-LC3 pAb

MBL international corporation

Upstream tips
- For Western Blot, lyse cells with RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF)
Protocol tips
- For Western Blot dilute primary Ab at 1:1000 and incubate at 4 °C overnight.

- For IF, dilute primary Ab between 1:500-:1000 and incubate at 4 °C overnight.
Atg5 (D5F5U) Rabbit mAb

Cell Signaling Technology

Upstream tips
- Lyse cells in 4X Laemmli buffer (0.125 M tris-HCl, pH 6.8, 4% SDS, 0.13mM bromophenol blue, 1 M sucrose) containing 0.5% ß mercaptoethanol
Protocol tips
- Dilute primary Ab at 1:1000

- Incubate primary antibody for 16 hours
Atg5 Antibody

Cell Signaling Technology

Upstream tips
- Lyse cells in RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF)
Protocol tips
-Dilute primary Ab at 1:1000 and at 4 °C overnight,
Anti-p62 (SQSTM1) (Human) pAb

MBL international corporation

Upstream tips
- Lyse cells in RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF)
Protocol tips
- Incubate primary antibody at 4 °C overnight
LC3A/B (D3U4C) XP® Rabbit mAb

Cell Signaling Technology

Upstream tips
- Lyse cells in RIPA buffer that contained 1% protease and phosphatase inhibitors
Protocol tips
- Dilute Ab at 1:1000

- Incubate primary antibody overnight at 4°C.
LC3B Antibody

Novus Biologicals

Upstream tips
- Lyse cells in RIPA buffer containing protease and phosphatase inhibitors
Protocol tips
- Dilute primary Ab between
1:500-1:3000 and incubate overnight at 4 °C.
SQSTM1/p62 (D1Q5S) Rabbit mAb

Cell Signaling Technology

Upstream tips
- Lyse cells with RIPA buffer that contained 1% protease and phosphatase inhibitors.
Protocol tips
- Incubate primary antibody overnight at 4°C.
Autophagy Assay Kit

Sigma-Aldrich

Upstream tips
- Seed 2 × 10^4 cells/well
Protocol tips
- Add reagent and incubate the cells at 37 °C with 5% CO2 for 15 minutes to 1 hour
SQSTM1 Antibody (D-3)

Santa Cruz Biotechnology

Protocol tips
- Dilute primary Ab between 1:100-1:1000

- Incubate primary antibody at 8°C overnight
Protocol tips
- Dilute primary Ab at 1:100

- Incubate primary antibody at 8°C overnight
APG5 Antibody (C-1)

Santa Cruz Biotechnology

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