Fluo 4 am

The intracellular Ca²⁺ concentration was measured using the Ca²⁺-sensitive fluorescent dye Fluo4-AM (Dojindo). The organoids cultured in hybrid hydrogel were washed with Ca²⁺-free Hanks’ balanced salt solution three times before being loaded with 5 μmol Fluo4-AM solution for 30 min at 37°C in the dark. The fluorescence was excited at 480 nm, and emission was detected between 510 and 550 nm. The images were captured with an Sp8 confocal microscope (Leica), and fluorescence intensity was analyzed in ImageJ.

C2-ceramide (Avanti Polar Lipid, Alabaster, AL, USA), TUDCA (Calbiochem, EMD-Millipore, Billerica, MA, USA), Tunicamycin, Thapsigargin, FB1, 4-PBA, S1P, Pluronic F-127 (Sigma-Aldrich, St Louis, MO, USA), and Fluo 4-AM (Dojindo Laboratories, Kumamoto, Japan) were reconstituted as recommended by their respective manufacturers. Antibodies against p-eIF2α (Ser51), eIF2α, JNK, cleaved caspase-3, p-ERK (Thr202/Tyr204), ERK (Cell Signaling Technology, Beverly, MA, USA), p-JNK (Thr183/Tyr185) (Abcam, Cambridge, MA, USA), caspase-3 (Abgent, San Diego, CA, USA), and actin (Santa Cruz, Dallas, TX, USA) were used in this study. All secondary antibodies were purchased from Abcam.

For in vitro experiments, [Ca²⁺]_i was monitored using Fluo-4 or R-GECO as previously described [8 (link)]. Briefly, C2C12 myotubes were cultured in a serum-free medium for 6 to 9 h and then cultured for an additional 2 h in a buffer containing 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES and 10 mM glucose (pH 7.0). Single fibers from soleus or EDL muscles were isolated by digestion using type 1 collagenase as previously described [9 (link),52 (link)]. Myotubes or isolated single fibers were incubated in 4 µM Fluo-4 AM (Dojindo, Rockville, MA, USA) for 30 min at room temperature to allow homogenous intracellular distribution of the dye. After incubation at 37 °C, the Fluo-4 loaded cells were placed on the stage of an inverted microscope (Olympus, Tokyo, Japan), and fluorescent intensity changes were recorded every 3 s. Data were calculated as normalized fluorescence ΔF/F0: ΔF/F0 = (Fmax − F0)/F0, where Fmax was the maximum fluorescence and F0 was the fluorescence before exposure to a reagent. Where noted, thapsigargin was combined with Fluo-4 AM, and the extracellular Ca²⁺ was depleted using a Ca²⁺-free buffer containing 2 mM EGTA. For in vivo Ca²⁺ imaging analysis, soleus or plantaris muscles were immediately dissected from pCAGGS-GCaMP2 mice before or after exercise, and the fluorescence was observed using a Biozero digital microscope (Keyence, Osaka, Japan).

Fluo 4-AM (F312, Dojindo, Japan), a Ca²⁺ -specific vital dye, was used to measure intracellular calcium levels. Dilute 1 mM Fluo 4-AM stock solution to 5 μM Fluo 4-AM working solution using HBSS buffer. The working solution (Fluo 4-AM) was incubated in cell incubator for 0.5 h. After washing the RAW264.7 cells three times with HBSS buffer, 1 ml of HBSS buffer was added to continue incubation for 30 min in a cell incubator. After the end, the content of calcium ions in each sample was detected according to the fluorescence intensity using a laser confocal microscope.

Two days after plating 2.5 × 10⁴ H295R cells into 96-well plates, the cells were incubated with and without 10 mM YM750 for 24 h. The cells were then loaded with Fluo 4-AM (Dojindo, Rockville, MD, USA; 5 mg/mL) in the presence of 1.25 mM probenecid (Dojindo) and 0.04% Pluronic F-127 (Dojindo) for 1 h. Cells were then washed with PBS and the recording medium containing 1.25 mmol/L probenecid and 20 mM KCl was added to the media. Changes in intracellular calcium concentration were determined by measuring the fluorescence intensity (excitation wavelength, 485 nm; emission wavelength, 535 nm).

Intracellular calcium assay was performed as previously described [30 (link)]. The culture medium of the Flp-In NCC HEK293 cells was replaced by loading buffer containing 5 μg/ml Fluo 4-AM (Dojindo, Kumamoto, Japan), 1.25 mmol/l probenecid (Dojindo, Kumamoto, Japan), and 0.02% Pluronic F-127 (Dojindo, Kumamoto, Japan). Following incubation with 5 mM EGTA or 1 μM SEA0400 in the loading buffer for 1h at 37°C, the loading buffer was replaced by recording medium containing 1.25 mmol/l probenecid. NCX1 siRNA silencing was applied 48 h before loading buffer replacement. Following the administration of KCl (final concentration: 10 mM), the fluorescence intensities of Fluo 4 were quantified from five regions of interest using LSM 510 Meta confocal microscopy and the Zen 2009 software (Carl Zeiss, Oberkochen, Germany).
http://dx.doi.org/10.17504/protocols.io.baihicb6