Fluo 3 am

The TRPV1 channel is a calcium ion channel, and it plays a crucial role in the flow of calcium ions into cells. The effect of this channel on calcium ions in cardiomyocytes was detected by Fluo-3 AM fluorescent probe method. The H9c2 cells were treated with a medium containing MA or various doses of triterpenoids for 24 h. Groups allocation and dose were the same as in Section 2.4.5. Following the treatment, the Fluo-3 AM (Solarbio, Beijing, China) (5 μmol/L) was used to stain the cells. The cells were then rinsed with HBBS after being incubated for 60 min at 5% CO₂ at 37°C, and the fluorescent intensity was assessed using a fluorescence microscope.

[Ca²⁺]i in lymphocytes were determined as previously reported [22 (link), 29 (link)]. Briefly, cells (1 × 10⁵) were incubated with Fluo-3/AM (5 μM) (Solarbio Co., Beijing, China) at 37°C for 30 min. After washing with PBS, cells were observed, and the images were acquired using a fluorescence microscopy equipped with an FITC filter (Nikon Ti-FL; Nikon Cooperation, Japan). For quantitative analysis, the fluorescent signals reflecting the [Ca²⁺]i level were measured by FCM, and the data were analyzed using FlowJo V10 software. Intracellular [Ca²⁺]i was reflected by Fluo-3 fluorescent intensity.

Cytoplasmic calcium assays were performed to detect intracellular calcium concentration after combination therapy (Liu et al., 2016 (link)). Briefly, overnight-cultured cells were washed and diluted with HBSS.D-Hanks buffer (Thermo Fisher, United States) (final concentration 1 × 10⁷ CFU/mL), and then mixed with 5 μM calcium indicator Fluo-3-AM (Solarbio, China) and 20% Pluronic F-127 (Meilun, China). The suspensions were incubated with agitation (200 rpm) at 35°C for 30 min, washed three times with HBSS buffer, and diluted to 1 × 10⁷ CFU/mL. After drug treatment, the cells were shaken at 35°C in the dark. Fluorescence was detected by inverted fluorescence microscopy and flow cytometry (Beckman, United States) at 0, 2, and 3 h.

The intracellular Ca²⁺ concentration was measured as previously described [42 (link)]. Briefly, PC12 cells were seeded on 6-well plates at a density of 1 × 10⁵ cells/well. At the end of the treatment, cells were incubated with 10 μM fluo-3/AM (Solarbio, China) for 40 min at 37 °C in the dark. Subsequently, the cells were resuspended with HEPES-buffered saline, and intracellular Ca²⁺ concentrations were determined by excitation and emission wavelengths at 506 nm and 526 nm, respectively. Furthermore, the fluorescence intensity of Ca²⁺ in PC12 cells was measured by laser scanning fluorescence microscopy.

Cells at a final concentration of 1 × 10⁵ cells/mL were seeded into 6-well plates and subjected to H/R intervention. Fresh kidney tissues were ground, and pre-cooled PBS washed 2 times. Single-cell suspension of 1 × 10⁵ cells/mL was prepared. For detection of intracellular Ca²⁺ concentration, 4 μL Fluo-3 AM (Solarbio, Beijing, China) was added to H/R NRK-52E cells and kidney single-cell suspension, and the Fluo-3 AM final concentration was 5 μM. For detection of mitochondrial Ca²⁺ concentration, 4 μL Rhod-2 AM (Thermo Scientific, MA, USA) was added to H/R NRK-52E cells and kidney single-cell suspension, and the Rhod-2 AM final concentration was 5 μM. For detection Ca²⁺ concentration in ER, 4 μL Mag-Fluo-4 AM (Thermo Scientific) was added to H/R NRK-52E cells, and the Mag-Fluo-4 AM final concentration was 5 μM. Cells were incubated at 37 °C for 30 min and washed twice with PBS. The incubation was continued by adding 2 mL PBS into the incubator at 37 °C for 30 min. Samples were collected, and 500 μL PBS was added to resuspend the cells for fluorescence intensity detection at excitation wavelength of 500 nm and emission wavelength of 525 nm using flow cytometry.