Bcecf am ph probe

Microscope-based PercevalHR ATP/ADP level detection was performed by using laser confocal microscopy. Briefly, 2 × 10⁴ cells were seeded in a 96-well clear bottom black plate (Costar, 3603). Images were taken by a Zeiss LSM880 confocal microscope using a Plan-Apochromat 20×/0.8 N.A. M27 objective at 37 °C. PercevalHR was excited using a 488 and 405 nm laser, for the MgATP-bound conformation and ADP-bound conformation, respectively. Emission was captured through a 450–550 nm filter. pHRed was excited using a 488- and 405-nm laser, and emission was captured through a 560 nm long pass filter. Four channel image sets were taken with no delay between individual channel acquisitions. For time-lapse studies involving PercevalHR, cells were imaged on IN Cell Analyzer 2500 HS high content analysis (HCA) imaging system using a 20×/0.8 N.A. objective. Time-series images were processed using Fiji. The investigators were blinded to sample allocation.
For flow cytometry, cells were collected and resuspended in HBSS. Ratios of FITC and KO525 channels of the CytoFLEX Platform (Beckman Coulter) were used to detect ATP/ADP levels. PH-correction was performed by using a BCECF-AM pH probe (DOJINDO, B262) according to the protocol provided by the producer.

The PercevalHR ATP/ADP level detection was performed using flow cytometry. Three dimensional cultured cells were harvested and re-suspended in HBSS. The ratios of the FITC and KO525 channels on the CytoFLEX Platform (Beckman Coulter) were used to detect ATP/ADP levels. PH-correction was performed using a BCECF-AM pH probe (DOJINDO, B262) according to the protocol provided by the manufacturer.