Quantifying Autophagosome-Lysosome Fusion in Adipocytes

Preadipocytes infected with RFP-GFP-LC3 adeno-associated virus were treated with 1 μM celastrol, 40 μM chloroquine or 200 nM bafilomycin A1 for 24 h. Cells were fixed with 4% paraformaldehyde and blocked with 5% BSA, then subjected to primary antibody LAMP1 (Cell Signaling Technology, United States, Cat #9091, RRID:AB_2687579, 1:100 dilution) overnight, followed by incubation with Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody (Abcam, United States, Cat # ab150079, RRID:AB_2722623, 1:500 dilution). Finally, the autophagosomes were observed under a fluorescence microscope (Leica DMI8, United States). Yellow against red puncta ratio was determined by the exact puncta numbers in five random fields of one slice obtained from YFP and RHOD channels separately. The Pearson correlation coefficient (PCC) for the colocalization of RFP-LC3 and Alexa Fluor 647-LAMP1 was calculated with the Coloc2 module of ImageJ (National Institutes of Health, United States, RRID:SCR_003070) (Elimam et al., 2019 (link)).