Fluo 8

Splenocytes isolated from C57BL/6 mice were treated with vehicle or DHT for 1 h. Cells were loaded with Fluo-8 (Abcam) and incubated for 30 min at 37 °C. Intracellular calcium flux was measured using flow cytometry. For the analyzing the response to anti-CD3 antibody, dye-loaded cells were recorded for initial 30 s and then monitored after addition of anti-CD3 antibodies, followed by ionomycin addition at 300^th sec.

4T1 cells were initially seeded at
a density of 4 × 10⁴ cells/well in eight-well chamber
slides (ibidi, Gräfelfing, Germany). After a 24-h incubation
period, the cells were subjected to various treatment conditions.
These treatments included media containing different components, including
free capsaicin (50 μM in 0.025% DMSO), free capsaicin+CPZ (15
μM), capsaicin prodrug (containing 50 μM of capsaicin)+GSH
(10 mM) or capsaicin prodrug+GSH+CPZ, for a duration of 2 h. Following
the respective treatment periods, the cells were carefully washed
three times with PBS. Subsequently, they were stained with a Ca²⁺ indicator, Fluo-8 (5 μM, Abcam, Cambridge, MA, USA),
for 30 min at 37 °C. After another three washes with PBS, the
fluorescence signals were observed using a fluorescence microscope
(Axio Observer 7, Carl Zeiss, Jena, Germany)

For intracellular calcium concentration measurement under fluid flow condition, 1 × 10⁵ MLO-Y4 cells were seeded in a µ-Slide I Luer (0.4 mm) fluid chamber slide (IBIDI, Germany) overnight. One hour before initiating fluid flow, the culture medium was removed and 100 µl Hank's Buffer with Hepes (HHBS) containing 4 µM Fluo-8 (Abcam, Cambridge, MA) was added to the culture, as described by the manufacturer. The cells were then cultured at 37°C for 30 min. After additional incubation at room temperature for 30 min, the chamber slide was placed under a confocal microscope in order to record the intensity of fluorescence of MLO-Y4 cells. Fluorescence was recorded for 3 min before starting fluid flow using HHBS and then recorded for another 10 min. The increase of intracellular concentration was calculated by subtracting the initial mean fluorescence. For measuring intracellular calcium concentration in cells with Yoda1 treatment, we cultured 4,000 MLO-Y4 cells per well in a 96-well-plate. We preloaded the cells with Fluo-8 as described by the manufacturer and read the intensity of the fluorescence using a Victor X3 multi-label plate reader (Perkin Elmer, Waltham, MA) immediately after the treatment. We measured the fluorescence for 5 min at an interval of 20 s. The percentage of increase in intracellular calcium concentration was calculated as (F_x-F₀)/F₀.

Sodium butyrate, SKF-96365, Pluronic^® F-127, Niacin, Mepenzolate bromide and LY-333531 hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Compound C and Fluo-8 were purchased from Abcam (Cambridge, UK). Permeant inhibitor of MLC kinase (PIK) was synthetized by Top-peptide (Shanghai, China) as described previously [25 (link)].

Jurkat cells were incubated with 1 μM (R)-9b for 3 h and 6 h and washed with PBS. Similarly, splenocytes isolated from C57BL/6 mice were treated with 1 μM (R)-9b for 6 h. Cells were loaded with Fluo-8 (Abcam) and incubated for 30 min at 37 °C. Intracellular calcium flux was measured using flow cytometry. Ionomycin was added at 300th second. Splenocytes and thymocytes isolated from WT and Ack1 KO mice were incubated with TRAMP-C2 cells for 6 h and washed with PBS. Splenocytes from C57BL/6 mice were isolated and treated with (R)-9b for 6 h, washed and incubated with TRAMP-C2 cells for 6 h. Intracellular calcium flux measurement was done as described above. PBMCs isolated from healthy donors, CRPC and mHSPC patients were incubated with 1 μM (R)-9b for 6 h, washed and calcium flux was measured as described above. For the analyzing the response of (R)-9b treated cells upon anti-CD3 addition, dye-loaded cells were recorded for initial 30 s and then monitored after addition of anti-CD3 antibodies, followed by ionomycin addition.