Autophagy assay cell type - K562 cells

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 9 matching solutions for this experiment

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream 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
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

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×).