4.3. Measurement of ER Calcium Levels in Hepatocytes

Calcium levels in ER were assessed by measuring the release of calcium from the ER after TG treatment as described previously [22]. Briefly, 5 × 10⁴ cells were seeded in a 24-well plate, and after adhering and extending, the cells were exposed to 4 μg/mL TM for 8 h or left untreated. Then, the cells were washed with HBSS buffer without calcium and magnesium ions three times, and incubated with Fluo-8 AM (Abcam, San Diego, CA, USA) for 30 min. After washing cells three times with calcium-free HBSS, steady-state cytosolic calcium levels were measured for 1 min at 23 °C. ER calcium store depletion was induced by 2 μM TG treatment and the calcium fluorescence was detected at Ex/Em 490/525 nm. Calcium release values were normalized to the background levels.