Calcium-switch assay was performed as described previously [28] (link). In brief, cells were plated in 60 mm dish with growth medium and cultured for overnight. Then, the cells were starved in growth medium containing 0.1% FBS for overnight and the medium was replaced with LCM (calcium-free DMEM medium, GIBCO Life Technologies, Grand Island, NY) for 4 h and 1.8 mM CaCl2 was added for the indicated time. For immunofluorescence, cells were plated onto glass coverslips (Fisher Scientific, Suwanee, GA) placed in 12-well plate, cultured for 24 h, starved, and calcium-switch was carried out as described above. Then, the cells were fixed in 100% methanol at −20°C for 7 min, immunostained, and photographed as described above.
Calcium free dmem medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Calcium-free Dulbecco's Modified Eagle Medium (DMEM) is a cell culture medium formulation that does not contain calcium. It is designed for specialized applications where the exclusion of calcium is required.
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2 protocols using calcium free dmem medium
1
Calcium-switch Assay for Cell Culture
2
Calcium Signaling Modulates Cell Migration
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To study the effect of calcium signaling pathways on the migration of RAW264.7 cells under gradient FSS, four pharmacological agents were employed.
Similar to our previous studies (17, 18) , the agents were incubated with cells before being exposed to FSS. In brief, 10 µM Gadolinium chloride (Gd, Sigma, USA) was supplied as the blocker of mechanosensitive cation channel (MSCC). The calcium stored in endoplasmic reticulum (ER) was depleted by 1 µM thapsigargin (TG, J&K Chemicals, China). 10 µM U-73122 (Darmstadt, Germany) was adopted to inhibit phospholipase C (PLC). For the above blocking tests, 10 min incubation of chemical reagents was applied. Calcium-free DMEM medium (Gibco, USA) was used to remove the extracellular calcium.
Similar to our previous studies (17, 18) , the agents were incubated with cells before being exposed to FSS. In brief, 10 µM Gadolinium chloride (Gd, Sigma, USA) was supplied as the blocker of mechanosensitive cation channel (MSCC). The calcium stored in endoplasmic reticulum (ER) was depleted by 1 µM thapsigargin (TG, J&K Chemicals, China). 10 µM U-73122 (Darmstadt, Germany) was adopted to inhibit phospholipase C (PLC). For the above blocking tests, 10 min incubation of chemical reagents was applied. Calcium-free DMEM medium (Gibco, USA) was used to remove the extracellular calcium.
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