Fluorodish cell culture dishes

For live cell microscopy, 2 × 10⁴ sGFP sender and 2 × 10⁴ αGFP receiver cells were seeded into 35 mm FluoroDish Cell Culture Dishes (World Precision Instruments, FD35-100) and immediately imaged under a DeltaVision OMX (GE Healthcare) microscope with a 60x oil objective lens (Olympus) in a humidified chamber at 37°C with 5% CO₂. One image was taken per minute for 3 hr. Images were collected with a cooled back-thinned EM-CCD camera (Evolve; Photometrics).
For fixed cell microscopy, sender and receiver cells were seeded at the ratios indicated in Supplementary file 2 with a total number of 1 × 10⁵ cells onto Neuvitro-coated cover slips (Thermo Fisher Scientific, NC0301187) in a 12-well cell culture plate. 24 hr after co-culture, cells were fixed in 4% paraformaldehyde (PFA) PBS solution at room temperature for 10 min and washed with PBS and distilled water three times each, before mounting onto slides using 50% glycerol. Images were captured using a Leica DMI6000B inverted microscope with an 40x oil objective lens. For quantification, GFP-containing receiver cells were counted. Multiple coverslips were analyzed across independent experiments (n = 10).

pH imaging experiments were performed using an inverted fluorescence microscope (Zeiss Axio Observer, AX10, Feldbach, Switzerland) with a 63x objective (Plan-Apochromat) and a CoolSnap HQ2 camera (Photometrics, Tucson, USA). The time resolution of the imaging part was ~ 440 ms. Images were recorded using the Metafluor software (Molecular Devices, Sunnyvale, USA). Briefly, cells were grown in Fluorodish cell culture dishes (World Precision Instruments) or on 15-mm diameter glass coverslips (VWR, Dietikon, Switzerland) and incubated prior to the experiment in Tyrode solution containing 10 μM of 5(6)-FAM SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester], mixed isomers (BIOTIUM, Chemie Brunschwig, Basel, Switzerland), a ratiometric dye able to sense pH changes (excitation 460/488 nm, emission 520 nm) for 15 min in an incubator (37°C, 5% CO₂). The amine-reactive succinimidyl ester form of the dye binds exclusively amine groups on cell surface proteins. The baseline of the 5(6)-FAM SE ratio measured during the perfusion of the Tyrode solution showed a certain degree of photobleaching. We corrected for the photobleaching using Origin PRO software (OriginLab Corp, Northampton, USA). After baseline correction, the 90–10% fall time of the fluorescence signal was determined to classify the time course of the pH changes.

PMG were cultured on fluorodish cell culture dishes (World Precision Instruments, China) to image live mitochondria and were treated with MitoTracker Green (300 nM, Thermo Fisher Scientific, USA) solution in the dark for 30 min (37 °C, 5% CO₂) to label mitochondria. Fresh complete medium was added after the cells were rinsed with preheated PBS. Real-time mitochondrial imaging was performed using confocal laser scanning microscopy (Leica, Germany) and mitochondrial morphology was analyzed by using ImageJ software [28 (link)].