Pultra hot

Lentiviral plasmid encoding GCaMP6s and mCherry were purchased from Addgene (Watertown, MA, USA; #80146, a gift from Dr. Darrell Kotton and pUltra-hot, and #24130, a gift from Dr. Malcolm Moore, respectively). Lentiviral plasmid encoding caspase-3 (Cas-3) indicator was engineered in the lab using the pUltra-hot lentiviral vector for bi-cistronic expression of mCherry and Cas-3 indicator (GANLS-DEVD-BNES, #50835, Addgene, a gift from Dr. Robert Campbell). Lentiviral plasmids encoding organelle-targeted Ca²⁺ indicators (R-CEPIA1er, G-CEPIA1er and CEPIA2mt) were a gift from Dr. Masamitsu Iino lab. Lentiviruses (LVs) were produced by transfection of HEK-293T cells (Cat. No. CRL-3216, American Type Culture Collection, ATCC, Manassas, VA, USA) using Lipofectamine 2000 (Cat. No. 11668-027, Thermo Fisher Scientific, Waltham, MA, USA). Supernatant containing viral particles was collected, cleared by centrifugation, and frozen at −80 °C for later usage.

In order to measure mitochondrial matrix pH, we used another ratiometric dye namely mito-SypHer, which was a gift from Nicolas Demaurex (Addgene plasmid # 48251). Mito-SypHer was cloned into the lentiviral expression vector pUltra-hot, which was a gift from Malcolm Moore (Addgene plasmid # 24130) (for details see Sec. 2.3). The respective virus was added to the cells resulting in an overexpression of SypHer located in the mitochondria, which was checked microscopically. Transduced cells were seeded and cultivated according to the FLIM protocol. Per experiment and per treatment, 100 cells were recorded, each being imaged with two different excitation wavelengths (405 and 488 nm). Emission was recorded at 525 nm. For calculation of the pH value corresponding to the emission ratio (405/488), nigericin-high potassium method was performed according to the BCECF calibration described above. For assessment of mitochondrial matrix pH in the amyloid precursor protein (APP)-overexpressing model and respective control cells, cells were cotransduced for MitoSypHer and APP/control vector. Subsequently, 20,000 cells were analyzed for MitoSypHer fluorescence intensity using FACS analysis (FACS Calibur). Calibration was performed using the nigericin-high potassium method mentioned above.