The CliniMACS system has been shown to provide CD133+ cells at up to 97% high purity and yields of up to 81%, 22 23 with passive depletion of unwanted cells.
ICRAC was activated by passive depletion of intracellular Ca2+ stores using the intracellular solution of 105 mmol/L Cs-glutamate; 10 mmol/L HEPES; 20 mmol/L 1,2-bis o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; 8 mmol/L MgCl2, pH 7.2.
In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced Ca2+ influx due to SOCE.
Neither I CRAC) activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 microg ml -1) heparin.
Inhibition of CRAC elicited by passive store depletion using EGTA (10 mM) in the pipette solution, was observed upon acute administration in a rapid and dose-dependent manner.
For passive store depletion the internal pipette solution included (in mM): 145 Cs methane sulphonate, 20 EGTA, 10 HEPES, 8 NaCl, 3.5 MgCl2, pH 7.2.
Consistent with inhibition of Orai channel activity, Pyr2, Pyr3 or Pyr6 substantially inhibited typical Orai downstream signalling events in RBL mast cells (NFAT activation and degranulation) activated by passive store depletion.
Remarkably, in endothelial cells, despite a robust increase in Ca2+ following Ca2+ re-addition, whole-cell Ca2+-release-activated inward current (ICRAC) following passive store depletion could only be convincingly shown in the absence of divalent cations (Abdullaev et al., 2008; Beech, 2009; Girardin et al., 2010).
The internal pipette solution for passive store-depletion contained (in mM) 3.5 MgCl2, 145 cesium methane sulfonate, 8 NaCl, 10 HEPES, 20 EGTA, pH 7.2.
HEK293 cells co-expressing STIM1 with either Orai1 or Orai3 were clamped in the whole-cell configuration employing 20 mM EGTA in the pipette solution for passive store-depletion.
Whilst passive Ca2+ store depletion and Ca2+ entry were stimulated by incubation with thapsigargin.
