Human STING is a proton channel (Live-cell pH Calibration Meaurements)

hTERT-immortalized BJ1 cells (ATCC CRL-2522) were transduced with lentiviral ratiometric reporters targeted to GALT constructed based on designs reported in Linders et al. <em>ACS Chem. Biol. </em>2022, with superecliptic pHluorin and mRuby3. Transduced cells were sorted based on mRuby3 expression using a Sony MA900 sorter. BJ1 SEP-mRuby3 cells were plated in 24-well glass-bottom plates (Greiner Bio-One) at 40,000 cells/well. After 48 hours, cells were stained for 45 minutes at 37°C with 0.5 µg/ml Hoechst 34580 (Thermo Fisher Scientific, cat. #H21486). Cells were then washed and incubated in Fluorobrite DMEM (Thermo Fisher Scientific, cat. #A1896701) medium supplemented with 10% FBS, 1% Pen-strep, and 1x GlutaMAX (Thermo Fisher Scientific, cat. #35050061). For pH calibration experiments, cells were treated with buffers at defined pH values supplemented with nigericin and valinomycin following manufacturer’s instructions (Thermo Fisher Scientific cat. # P35379). Additional buffers at pH 7, 6, and 5 were created by titration with acid. All images were acquired using a Ti2-E inverted epifluorescence microscope (Nikon) with automated XYZ stage control, hardware autofocus, and a Yokogawa CSU-W1 confocal spinning disk unit with Zyla 4.2 PLUS sCMOS camera. An Okolab cage incubator was set to 37°C with 5% CO2. 405, 488, 561, and 640 nm laser lines were used for fluorescence illumination and all hardware was controlled using NIS elements software. Images were acquired using a 40X 0.95 NA CFI Plan Apo λ objective (Nikon MRD70470) with the following lasers and filters: Hoechst (405 nm laser, Chroma Multi LED set #89401), superecliptic pHluorin (488 nm laser, Chroma Multi LED set #89401), and mRuby3 (561 nm laser, Chroma Multi LED set #89401), assaying three z planes per field of view with 1.25 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection. Images are maximum projections of multiple z-stacks with each frame representing one pH value: 7.5, 7, 6.5, 6, 5.5, 5, and 4.5 (except for replicate 1, which omitted pH 7). Channels are: Hoechst 34580, SEP (super-ecliptic GFP), mRuby3, and SEP/mRuby3 (ratio). Crops indicate cropped fields of view presented in the manuscript.本数据集使用的细胞为hTERT永生化BJ1细胞（ATCC CRL-2522），通过慢病毒转导引入靶向GALT的比例型报告基因载体；该报告基因基于Linders等人2022年发表于《ACS化学生物学（ACS Chem. Biol.）》的设计构建，融合了超折叠pHluorin（superecliptic pHluorin）与mRuby3。转导后的细胞借助索尼MA900流式细胞分选仪（Sony MA900），基于mRuby3的表达水平完成分选。将转导得到的BJ1 SEP-mRuby3细胞以4×10⁴个/孔的密度接种于24孔玻璃底培养板（Greiner Bio-One）中。细胞培养48小时后，于37℃下用0.5 μg/mL的Hoechst 34580（赛默飞世尔科技，Thermo Fisher Scientific，货号H21486）染色45分钟。弃去染色液并洗涤细胞，随后将细胞置于添加了10%胎牛血清（FBS）、1%青霉素-链霉素（Pen-strep）及1×GlutaMAX（赛默飞世尔科技，Thermo Fisher Scientific，货号35050061）的Fluorobrite DMEM培养基（赛默飞世尔科技，Thermo Fisher Scientific，货号A1896701）中孵育。针对pH校准实验，按照赛默飞世尔科技（Thermo Fisher Scientific，货号P35379）的产品说明书操作，将细胞用添加了尼日利亚菌素（nigericin）与缬氨霉素（valinomycin）的特定pH值缓冲液处理；另通过酸滴定制备pH7、6、5的缓冲液备用。所有成像实验均采用尼康（Nikon）Ti2-E倒置落射荧光显微镜完成，该系统搭载自动XYZ载物台控制模块、硬件自动对焦单元，搭配Yokogawa CSU-W1共聚焦转盘单元及Zyla 4.2 PLUS sCMOS相机。实验环境通过Okolab笼式培养箱（Okolab）维持在37℃、5% CO₂条件。成像采用40× 0.95 NA CFI Plan Apo λ物镜（尼康MRD70470），搭配如下激光与滤光套装：Hoechst 34580（405 nm激光，Chroma Multi LED套装#89401）、超折叠pHluorin（488 nm激光，Chroma Multi LED套装#89401）及mRuby3（561 nm激光，Chroma Multi LED套装#89401）；每个视场采集3个Z平面，层间距为1.25 μm。视场通过NIS Elements成像软件（NIS Elements）的坐标自动选取，无需人工预筛选。405、488、561及640 nm激光线用于荧光激发，所有硬件设备均通过该软件控制。最终图像为多张Z堆叠的最大强度投影图，每个图像帧对应一个pH值：7.5、7、6.5、6、5.5、5及4.5（其中重复实验1除外，该组未采集pH7的图像）。成像通道包括：Hoechst 34580通道、SEP（超折叠GFP，super-ecliptic GFP）通道、mRuby3通道及SEP/mRuby3比例通道。文中展示的裁剪区域即为本数据集的视场裁剪图。