Intracellular ph assay kit

Intracellular pH was measured according to the protocol of the fluorometric Intracellular pH Assay Kit (Sigma, Schnelldorf, Germany, MAK-150). Fluorescent BCFL-AM indicator that was able to pass through the cell membrane was used to measure intracellular pH fluctuations in the fluorometric intracellular pH kit. BCFL-AM was utilised to measure the reductions in intracellular pH of the cells that had been treated by various conditions. The cells were plated on black plates, as 6 × 10⁴ in each well of the plate, and incubated for 24 h. The medium was replaced with BCFL-AM Reagent which had been prepared in 100 µl of HBS solution (Hank’s buffer with 20 mM HEPES, 5 mM Probenecid), then the cells were incubated at 37 °C in an atmosphere of 5% CO₂ for 30 min (protected from light). Sulfonamide compound at doses of 0–25 μM was added to the HBS solution. In the next 5 min, the measurement was performed at wavelengths of 490 nm (excitation) 535 nm (emission) in spectrofluorimetry (Spectramax, M5). Extracellular pHe and lactate levels were measured with commercial kit by the blood gas device (ABL90 FLEX PLUS, Radiometer, Copenhagen, Denmark) which is routinely used in the clinical biochemistry laboratory of hospital. After the application of the substance in this method, the media were analysed directly by the device.

Intracellular PH was measured according to the protocol of fluorometric Intracellular pH Assay Kit (Sigma-Aldrich, MAK-150, Germany). Fluorescent BCFL-AM indicator passed through cell membrane to measure intracellular pH fluctuations in the fluorometric intracellular pH kit. BCFL-AM was utilized to measure intracellular pH of the cells. The cells were planted in black plates and incubated for 24 h. The medium was replaced with BCFL-AM Reagent, which had been prepared in 100 µL of HBS solution (Hank’s buffer with 20 mM HEPES, 5 mM Probenecid); then, the cells were incubated at 37 °C with an atmosphere of 5% CO₂ for 30 min (protected from light). The compound E at doses of 0–200 μM was administrated in HBS solution. The measurement was performed at wavelengths 490 nm (excitation) 535 nm (emission) in fluorimetry (Spectramax M5, BioTek, VT, USA.