Code for analysing intracellular pH on a single cell level

Cells were plated in triplicate at 50,000-100,000 cells per well in black wall, flat coverslip bottom µ-plate 96-well plates with a growth area of 0.56 cm2 per well (Ibidi) and were left to attach overnight. They were then incubated in media supplemented with cSNARF1-AM (5 µg/mL, Invitrogen) and the nuclear stain Hoechst-33342 (10 µg/mL, Molecular Probes), for 15 min, and then replaced with medium of varying sodium bicarbonate concentration (twice). Images of fluorescence excited at 377 nm and collected at 447 nm (Hoechst-33342), and of fluorescence excited at 531 nm and collected at 590 nm and 640 nm (cSNARF1), were acquired using Cytation 5 imaging plate reader (Biotek) and its bespoke software. Images were either acquired using a 4x objective, or a 10x objective, as indicated in figure legends. Measurements were performed in an atmosphere of 37 °C and 5% CO2, established in the plate reader. Further analysis of the population distribution of pH data was performed using a MATLAB script. Images saved as stack.tif act as input for the script (supplementary_code_1). cSNARF1 fluorescence ratios were converted into pHi using a calibration curve obtained through the nigericin method. pHi distributions from replicate wells were pooled, and low intensity measurements were removed using a second MATLAB script (Supplementary_Code_2).

将细胞以每孔50000~100000个的密度接种至3个重复孔中，所用耗材为底部带有平整盖玻片的黑色壁式96孔µ-plate培养板（Ibidi），单孔生长面积为0.56 cm²，接种后静置过夜以完成贴壁。随后将细胞置于添加了cSNARF1-AM（5 μg/mL，Invitrogen）与细胞核染料Hoechst-33342（10 μg/mL，Molecular Probes）的培养基中孵育15分钟，之后更换为不同碳酸氢钠浓度的培养基，该更换操作重复两次。采用Cytation 5成像微孔板读数仪（Biotek）及其定制软件，采集激发波长377 nm、发射波长447 nm处的荧光图像（对应Hoechst-33342染色），以及激发波长531 nm、分别在590 nm与640 nm处采集的荧光图像（对应cSNARF1染色）。图像采集分别采用4倍物镜或10倍物镜，具体配置详见图例说明。所有检测均在微孔板读数仪内置的37℃、5% CO₂环境中完成。采用MATLAB脚本对pH数据的群体分布进行进一步分析，以stack.tif格式保存的图像作为该脚本的输入文件（supplementary_code_1）。通过尼日利亚菌素（nigericin）法构建校准曲线，将cSNARF1的荧光比值转换为细胞内pH（pHi）值。合并重复孔的细胞内pH分布数据，并通过第二份MATLAB脚本（Supplementary_Code_2）剔除低强度信号的测量值。