Ph calibration buffer kit

To estimate how nanoCaCO₃ were affecting the tumor microenvironment, pH measurements were made using 24 well plates. First, MDA-MB-231 cells were seeded in the wells at a concentration of 3.0 × 10⁶ cells mL⁻¹. The next day, the media was replaced with either plain media (control) or media containing 0.8 mg mL^-1 nanoCaCO₃ (experimental). After 3 more days, the media was collected, and a pH probe (AB15 Basic, ThermoFisher) was used to measure the pH of the media in the wells.
Additionally, the intracellular pH was determined using a pHrodo Green AM Intracellular pH Indicator (ThermoFisher) following the manufacture’s protocol. With this dye, higher fluorescent intensities indicate a lower pH. Therefore, to correlate fluorescent intensities with pH values, a pH Calibration Buffer Kit (ThermoFisher) was used.

Endosomal pH measurements were conducted using our previously published protocols.10 Briefly, U251n cells plated in fluorodishes (World Precision Instruments) were placed on ice for 10 minutes and then rinsed with cold imaging buffer (Live Cell Imaging Solution (Thermo Fisher Scientific) with 20 mmol/L glucose and 1% BSA) to remove residual serum transferrin. Cells were then incubated with 50 µg/mL pH‐sensitive transferrin (fluorescein‐conjugated transferrin, Tfn‐FITC; Thermo Fisher Scientific), in imaging buffer for 30 minutes. LCIS was used to rinse the cells, following which fluorescence images were acquired (excitation 494 nm and emission 518 nm) with Lumascope 620 (Etaluma). Internal fluorescence was quantified using ImageJ 15 software, and average fluorescence intensity was recorded. NHE9‐mcherry was transfected using Lipofectamine 2000 for expression in U251n cells. Tfn‐FITC fluorescence was quantified only in mcherry‐positive cells. To normalize for total transferrin uptake, pH‐insensitive transferrin (50 µg/mL Alexa Fluor 568‐conjugated transferrin (Tfn‐568) was loaded. A pH calibration buffer kit (Thermo Fisher Scientific) was used to generate a standard curve from which endosomal pH was determined.

After trypsinzing and collecting the cells, the lysosomal pH value of BMDM-M1, BMDM-M2, and naphplatin-treated BMDM-M2 cells was measured by staining with LysoSensor Yellow/Blue DND-160 (1:1,000 diluted and pre-incubated in complete culture medium for 30 min at 37 ℃. Thermo Fisher Scientific, L7545) for 3 min at 37 ℃. Cells were transferred into a black 96-well plate (1 × 10⁶ cells per 200 μl per well) after being washed with PBS. The fluorescence intensity was measured on the Synergy H1 (BioTek) at Ex-360/Em-440 and Ex-360/Em-550 with 10 μM of valinomycin and 10 μM of nigericin was added. We used the intracellular pH Calibration Buffer Kit (Thermo Fisher Scientific, P35379) to measure the standard curve of lysosomal pH value. For LysoSensor Green probes (Thermo Fisher Scientific, L7535) staining in BMDM-M1 and M2 cells, cells were incubated with probes for 30 min to 1 hour, then the PBS was used to collect and wash the cells to measure the mean fluorescence intensity by flow cytometry.