Lyso tracker red fluorescent probe

Lysosomes or mature autophagosomes were detected with Lyso-Tracker Red fluorescent probe (Beyotime Biotechnology). Lyso-Tracker Red penetrates mMECs and detects lysosomes or mature autophagosomes in an acidified environment [33 (link)]. In these experiments, mMECs were seeded in 6-well plates with coverslips and challenged with S. uberis at 80% confluence. Cells were washed thrice with ultra-sterilized PBS and incubated in 5% CO₂ at 37 °C for 30 min with 50 nM Lyso-Tracker Red in cell culture medium for lysosome red staining. The Lyso-Tracker Red was removed by three washes with PBS and nuclei stained with DAPI for 5 min. Fluorescent signals were observed under a laser scanning confocal microscope at wavelengths of 405, 488 and 561 nm.

The microtubule associated protein light chain 3 (LC3) protein is a membrane marker of autophagosome. Therefore, autophagosomes formation was detected by detecting the GFP fluorescence intensity of cells transduced with the fused GFP-LC3 gene. H9C2 cells were seeded at a concentration of 4 ×10⁴ cells/well in 24-well plate, and then Ad-GFP-LC3b (C3006, Beyotime, China) adenovirus transfection was performed followed by adherent culture for 24 h. After transfection for 24 h, the corresponding cultures were replaced.
Lysosomes were detected using the Lyso-Tracker Red fluorescent probe (C1046, Beyotime), an eosinophilic fluorescent probe for the labelling and tracing of acidic organelles within living cells. According to the manufacturer’s instruction, the cell culture medium was removed, the prepared Lyso-Tracker Red staining working solution was added and co-incubated with the cells for 60 min at 37° C. The Lyso-Tracker Red staining working solution was removed and fresh cell culture medium was added, followed by observation with a fluorescent microscope.