2.8. Measurement of autophagic influx

Autophagy flux in cardiac fibroblasts was measured using a plasmid expressing mCherry and GFP-tagged LC3 (pBABE-puro mCherry-EGFP-LC3B; Addgene, 22,418). Cardiac fibroblasts grown on 4-well chamber slides were transfected using Lipofectamine® 3000 Reagent from Invitrogen. 24 h after transfection, the cells were treated with 2.5 μg/ml recombinant human chymase, 5 μM rapamycin, and 100 μM chloroquine for 2 h followed by3%formaldehyde fixation prior to image acquisition as described above. The number of red and green punctate in at least 40 transfected cells in each group were counted using Image Pro Software (MediaCybernetics®). The progression of autophagy is started with the formation of autophagosomes shown as yellow punctate vesicles because they are marked with both red and green fluorescence. When the autophagosomes fuse with the lysosomes to form autolysosomes, the GFP green fluorescent signal is lost because the acidic environment quenches the GFP fluorescence. The augmented appearance of red-only labeled autolysosomes is an indication of the autophagic flux.