Macrophages were incubated with ACs labeled with PKH26 (Thermo Fisher), according to the manufacturer’s protocol. The macrophages were then rinsed twice with PBS and incubated for 30 minutes with 5 μM Fluo-8-AM (Abcam) in a humidified CO2 incubator at 37 °C. The macrophages were then rinsed with PBS twice, followed by live cell imaging on a Nikon spinning disk confocal microscope (Eclipse Ti) using Nikon NIS-Elements software to quantify the MFI of the Fluo-8-AM signal in AC− and AC+ macrophages.
Fluo 8 am
Manufactured by Nikon
Sourced in Japan
Fluo-8-AM is a fluorescent calcium indicator dye used for monitoring intracellular calcium levels. It is a cell-permeant acetoxymethyl (AM) ester form of the Fluo-8 fluorophore, which exhibits a significant increase in fluorescence upon binding to calcium ions.
Automatically generated - may contain errors
2 protocols using fluo 8 am
1
Macrophage Phagocytosis and Calcium Flux
2
Measuring Calcium Signaling in HLEpiCs
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The [Ca 2+ ] i was determined as previously described [23] . Brie y, HLEpiCs were loaded with the green uorescent calcium-binding dye Fluo-8/AM (10 µmol/L; abcam) and 0.02% pluronic acid and placed in a dark incubator for 1 h at 37 °C. The Ca 2+ release was activated by 100 µmol/L ATP (Sigma) in Ca 2+ -free PBS that contained (in mmol/L) 140 NaCl, 5 KCl, 1 CaCl 2 , 1 MgCl 2 , 10 glucose, 0.2 EGTA, and 5 HEPES (pH 7.4). After the baseline stabilized, SOCE was detected by adding 2 mmol/L CaCl 2 . The [Ca 2+ ] i was recorded using uorescence microscopy (Nikon, Japan) because the uorescence intensity of Fluo-8/AM increases when it binds to Ca 2+ . Changes in the [Ca 2+ ] i were calculated as the ratio of uorescence intensities before (F 0 ) and after (F 1 ) adding CaCl 2 (F 1 /F 0 ).
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