Cytosolic Free Ca2+ Quantification

To measure relative levels of cytosolic free Ca²⁺, cells were stained with 3 mmol/L Fluo-4-AM for 25 min, washed twice with Dulbecco phosphate-buffered saline (DPBS), resuspended in DPBS without Ca²⁺, Mg²⁺ (if not otherwise indicated) plus 20 mmol/L HEPES and maintained at 37C while treating the cells as indicated and analyzing by BD FACSVerse flow cytometer (FL-1 channel).

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Liu B., Chen R., Zhang Y., Huang J., Luo Y., Rosthøj S., Zhao C, & Jäättelä M. (2023). Cationic amphiphilic antihistamines inhibit STAT3 via Ca2+-dependent lysosomal H+ efflux. Cell Reports, 42(2), 112137.

Publication 2023

BD FACSVerse flow cytometer

Cells Cytosolic Fluo 3 Hepes Phosphate Saline

Corresponding Organization : University of Copenhagen

Other organizations : Ocean University of China, BGI Group (China), Aarhus University

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Variable analysis

independent variables

Treating the cells as indicated

dependent variables

Relative levels of cytosolic free Ca2+

control variables

Fluo-4-AM concentration (3 mmol/L)
Staining duration (25 min)
Washing procedure (twice with DPBS)
Resuspension buffer (DPBS without Ca2+, Mg2+ plus 20 mmol/L HEPES)
Temperature (37°C)
Flow cytometry analysis (BD FACSVerse, FL-1 channel)

controls

Negative control: Not explicitly mentioned
Positive control: Not explicitly mentioned

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