Autophagy assay cell type - A375 (human melanoma cell line)

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 6 matching solutions for this experiment

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.
Protocol tips
- incubate primary Ab with agitation for 1 hour.
Downstream tips
- If high background or Non-specific bands is present,Try different blocking strategies: Longer blocking times, higher % of blocker, a different blocker, inclusion of blocker protein in antibody dilutions or try a fresh batch of the same blocker.
Upstream tips
- Do not freeze component A
Protocol tips
- Incubate the cells overnight (≥16 hours) after adding LC3 reagent.

- For best results, allow cells 48 hours post-transduction for expression levels to equilibrate
LC3A/B (D3U4C) XP® Rabbit mAb #12741

Cell Signaling Technology

Protocol tips
- diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween 20 at 4°C with gentle shaking, overnight.

- Dilute Primary Ab at 1:1000
Upstream tips
- Lyse cells using RIPA mixed with protease inhibitors and phosphatase inhibitors
Protocol tips
- Dilute Primary Ab at 1:500-1:3000
Protocol tips
- 1:500 dilution of primary antibody and overnight incubation at 4°C
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