FACS-based calcium mobilization assay
Several agonists induce the rapid increase in cytosolic free calcium (Ca2+) concentration upon cell activation. E.g. the chemokines CXCL8 and CXCL7 cause Ca2+ release from intracellular pools and influx through the chemokine receptor CXCR2. In our work, we have identified the chemokine receptor CXCR2 as a functional receptor for the chemokine-like cytokine MIF, which acts as a non-cognate ligand of CXCR2 (ref. 1). As MIF binding to CXCR2 resulted in typical chemokine effects such as cell arrest and chemotaxis, we also investigated the Ca2+ mobilization as an early signal of MIF-induced cell activation.
Neutrophils (obtained from healthy donors)
Fluo-4 AM
Assay buffer:
(130 mM NaCl, 4.6 mM KCl, 1 mM CaCl2, 5 mM glucose, 20 mM HEPES, pH 7.4)
In order to record the change of cytosolic Ca2+ concentration over time, we employed the FACSAria System (BD Biosciences, San Jose, CA).
2.5 h
Pass cells through separation filters to avoid clogging.
As the Ca2+ concentration increases within seconds after addition of the stimulus, it is important to start the measure as fast as possible.
Neutrophils are often prepared in buffers containing EDTA, which keeps them from agglutinating. Be sure that all EDTA is removed, before starting the experiment.
See Bernhagen et al., Nat. Med. 2007.
Posted 18 Apr, 2007
FACS-based calcium mobilization assay
Posted 18 Apr, 2007
Several agonists induce the rapid increase in cytosolic free calcium (Ca2+) concentration upon cell activation. E.g. the chemokines CXCL8 and CXCL7 cause Ca2+ release from intracellular pools and influx through the chemokine receptor CXCR2. In our work, we have identified the chemokine receptor CXCR2 as a functional receptor for the chemokine-like cytokine MIF, which acts as a non-cognate ligand of CXCR2 (ref. 1). As MIF binding to CXCR2 resulted in typical chemokine effects such as cell arrest and chemotaxis, we also investigated the Ca2+ mobilization as an early signal of MIF-induced cell activation.
Neutrophils (obtained from healthy donors)
Fluo-4 AM
Assay buffer:
(130 mM NaCl, 4.6 mM KCl, 1 mM CaCl2, 5 mM glucose, 20 mM HEPES, pH 7.4)
In order to record the change of cytosolic Ca2+ concentration over time, we employed the FACSAria System (BD Biosciences, San Jose, CA).
2.5 h
Pass cells through separation filters to avoid clogging.
As the Ca2+ concentration increases within seconds after addition of the stimulus, it is important to start the measure as fast as possible.
Neutrophils are often prepared in buffers containing EDTA, which keeps them from agglutinating. Be sure that all EDTA is removed, before starting the experiment.
See Bernhagen et al., Nat. Med. 2007.
© Research Square 2020