Autophagy assay cell type - Human osteosarcoma cancer 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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The Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	To monitor autophagosome maturation, i.e., traffic of autophagosomes and fusion with lysosomes (also called autophagic flux), MDA-MB-231 cells were transfected as described in 6-well plates with 600 ng of a plasmid expressing full-length APC (WT or m4). 12-16 h after transfection, the medium was replaced with serum-free media (Thermo Fischer Scientific) supplemented with 20 mM HEPES (pH 7.4) and 20 mM Lglutamine. Cells were then mixed with Premo™ Autophagy Tandem Sensor RFP-GFPLC3B (#P36239; Thermo Fischer Scientific) and incubated for 16 h as described in manufacturer’s instructions.

Protocol tips
To monitor autophagosome maturation, i.e., traffic of autophagosomes and fusion with lysosomes (also called autophagic flux), MDA-MB-231 cells were transfected as described in 6-well plates with 600 ng of a plasmid expressing full-length APC (WT or m4). 12-16 h after transfection, the medium was replaced with serum-free media (Thermo Fischer Scientific) supplemented with 20 mM HEPES (pH 7.4) and 20 mM Lglutamine. Cells were then mixed with Premo™ Autophagy Tandem Sensor RFP-GFPLC3B (#P36239; Thermo Fischer Scientific) and incubated for 16 h as described in manufacturer’s instructions.

Protocol tips

To monitor autophagosome maturation, i.e., traffic of autophagosomes and fusion with
lysosomes (also called autophagic flux), MDA-MB-231 cells were transfected as
described in 6-well plates with 600 ng of a plasmid expressing full-length APC (WT or
m4). 12-16 h after transfection, the medium was replaced with serum-free media
(Thermo Fischer Scientific) supplemented with 20 mM HEPES (pH 7.4) and 20 mM Lglutamine. Cells were then mixed with Premo™ Autophagy Tandem Sensor RFP-GFPLC3B (#P36239; Thermo Fischer Scientific) and incubated for 16 h as described in
manufacturer’s instructions.

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3 antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Cells were lysed in WB/IP lysis buffer (P0013, Beyotime), all the procedures were following the manufacturer's protocol. Subsequently the cell lysates were boiled in 5X SDS-PAGE loading buffer for 10 min and then resolved by 8% SDS-PAGE and transferred to nitrocellulose membrane. The following antibodies were used in this study: LC3 (Sigma), Beclin 1 (CST), PINK1 (CST), Parkin (CST), GAPDH (Proteintech). Bound antibodies were visualized with the ECL kit (Thermo Scientific).

Protocol tips
Cells were lysed in WB/IP lysis buffer (P0013, Beyotime), all the procedures were following the manufacturer's protocol. Subsequently the cell lysates were boiled in 5X SDS-PAGE loading buffer for 10 min and then resolved by 8% SDS-PAGE and transferred to nitrocellulose membrane. The following antibodies were used in this study: LC3 (Sigma), Beclin 1 (CST), PINK1 (CST), Parkin (CST), GAPDH (Proteintech). Bound antibodies were visualized with the ECL kit (Thermo Scientific).

Protocol tips

Cells were lysed in WB/IP lysis buffer (P0013, Beyotime), all the procedures were following the manufacturer's protocol. Subsequently the cell lysates were boiled in 5X SDS-PAGE loading buffer for 10 min and then resolved by 8% SDS-PAGE and transferred to nitrocellulose membrane. The following antibodies were used in this study: LC3 (Sigma), Beclin 1 (CST), PINK1 (CST), Parkin (CST), GAPDH (Proteintech). Bound antibodies were visualized with the ECL kit (Thermo Scientific).

Manufacturer protocol Publication protocol 1 Relevant paper

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