Pcmv lyso phluorin

Human cervical cancer HeLa cells were maintained in RPMI-1640 medium (Gibco, Paisley, UK), supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 μg/ml penicillin and streptomycin (Biological Industries, Beth-Haemek, Israel) in a humid atmosphere containing 5% CO₂ at 37°C. HeLa cells were transiently transfected using Linear Polyethylenimine (PEI, MW 25,000) transfection reagent (Polysciences, Pennsylvania, USA) at a ratio of 3 μg PEI : 1 μg DNA. For stable transfection with LAMP1-mCherry, 24 hr after transfection, cells were subjected to G-418 selection (400 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) in the growth medium. pLAMP1-mCherry was a gift from Amy Palmer (Addgene plasmid # 45147). pCMV-lyso-pHluorin was a gift from Christian Rosenmund (Addgene plasmid # 70113). pTurbo-RFP-C was from Evrogen (Moscow, Russia).

pHluorin imaging was carried out in HEK293T cells cotransfected with lyso-NpHR3.0/lyso-ArchT and pCMV-lyso-pHluorin (Addgene, #70113). All experiments were performed 24 h after transfection. lyso-NpHR3.0/lyso-ArchT was stimulated at 594 nm, while pHluorin fluorescence was excited at 488 nm by laser.
pHrodo dextran was used to monitor lysosomal pH changes in lyso-ChR2-expressing cells (Fig 2g–2l). Cells were incubated with pHrodo dextran (10 μg/ml; Thermo Fisher Scientific) for 6 h at 37°C and then cultured in medium without pHrodo dextran for an additional 1 h. Bafilomycin A1 (1 μM, MCE) was used to inhibit V-ATPase (Fig 2c, 2d, 2i and 2j). Imaging was performed with a Plan-APOCHROMAT 100×/1.4 Oil DIC oil immersion objective on an LSM980 confocal microscope (Zeiss).