| Assay Method Information | |
| | Erythrocyte KCa3.1 Assay |
| Description: | Human blood was drawn from healthy human volunteers in a standard heparinized blood sampling vial (Vacutainer, Li/heparin, ED Bioscience, Plymouth, UK). The erythrocytes were packed by centrifugation, and the plasma and buffy coat were removed by aspiration. Erythrocytes were washed three times in the experimental salt solution and then stored at 0 C. until use. Blood samples from NMRI mice or from Wistar rats were treated similarly. The methodological principle is outlined in Macey et al. (1978) and further described in Str baek et al. (2013). Activation of the erythrocyte KCa3.1 channels were obtained by addition of the Ca2+ ionophore A23187, which causes synchronized hyperpolarization, which is reported as a CCCP-mediated shift in the unbuffered extracellular pH of the erythrocyte suspension. Standard procedure: 3 mL unbuffered experimental salt solution (in mM: 2 KCl, 154 NaCl, 0.05 CaCl2) was heated to 37 C. with stirring. Packed erythrocytes were added (50 μL, final cytocrit 1.5%), and the extracellular pH (pHo) followed with a glass/calomel (pHG200-8/REF200, Radiometer, Denmark) electrode pair. CCCP (3 μL, final concentration 20 μM) was added followed by varying concentrations of test compounds (DMSO concentration constant). After pH stabilization at 7.2, A23187 (3 μL, final concentration 0.33 μM) was added to initiate the experiment. After the peak hyperpolarization was attained, the intracellular pH (pHi constant during the experiment) was found by haemolysing the erythrocytes via addition of 100 μL of Triton-X100. |
| Affinity data for this assay | |
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