Viability cytotoxicity kit

For the cell tracker experiments, astrocytes and LUHMES cells were incubated, respectively, with green cell tracker CMFDA (10 ​μM, product number C7025, Invitrogen) and red cell tracker CMTPX (10 ​μM, product number C34552, Invitrogen) for 30 ​min in PBS at 37 ​°C with 5% CO₂ and then washed three times with cell culture media. For the image series in Fig. 4, the astrocytes were re-stained just before seeding LUHMES cells to obtain a stronger fluorescent signal to counter the dilution of the fluorophore due to the proliferation of the astrocytes. The green and red colors of the dyes were replaced in Fig. 4 with a blue/red colorblindness-friendly palette. For the calcium imaging, cells were incubated in 0.75 ​μM Calbryte™ 520 AM (AAT Bioquest, product number 2065) for 45 ​min at 37 ​°C and washed three times with respective cell media before the experiment. For the live/DEAD assay, we used a Viability/Cytotoxicity Kit (product number L23224, Invitrogen). Samples were incubated in a final concentration of 2 ​μM calcein AM and 4 ​μM Ethidium homodimer-1 in PBS for 30 ​min at RT. All live imaging was performed with a Zeiss Cell Observer microscope equipped with an incubator chamber, heating, CO₂ control, and a Zeiss AxioCam MRm camera.

For determine the cytotoxicity of felodipine at its MIC level, the viability of HepG2 (ATCC HB-8065), human keratinocytes (HaCaT) and rat bone marrow mesenchymal stem cells (rBMSC) were examined as previously described [[32] (link), [33] , [34] (link)]. Briefly, 100 μL of HepG2, HaCaT and rBMSC cells were inoculated into a 96-well plate at a density of 1.0 × 10⁴/well and incubated at 37 °C 5% CO₂ for 24 h to allow cells to adhere to the plate. Next, a 2-fold serial dilution of felodipine (ranging from 64 μg/ml to 0 μg/ml) were added to the 96-well plates and incubated for another 24 h. Then, the viability of mammalian cells was examined using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) and the Viability/Cytotoxicity Kit (Invitrogen, CA, USA) following the manufacturer's instructions. Fluorescence microscopy (Nikon, Japan) was used to visualize the labeled cells. Live cells were stained with Calcein-AM (green), while dead cells were stained with Ethidium homodimer-1 (EthD-1; red).