Measurement of Ca²⁺ release and Ca²⁺ influx in HL-60 cells treated with tBHQ.

SERCA enzyme inhibition-induced Ca²⁺ mobilization and ensuing capacitative calcium influx were measured spectrofluorimetrically using Fura-2–loaded HL-60 cells. (A) Cells were treated as described in “Materials and methods,” and Ca²⁺ release from sensitive intracellular pools was measured in cells suspended in Ca²⁺-free extracellular medium immediately after addition of 12 μM tBHQ (first peak). Thereafter, following addition of Ca²⁺, capacitative Ca²⁺ influx was recorded (second peak). To compare the magnitude of calcium release or influx obtained at various concentrations of tBHQ, the area under the calcium peaks was determined using the NIH Image 1.62b7 software (National Institutes of Health, Bethesda, MD) and is expressed as percentage of the maximal value (100%) obtained using the maximally effective concentration of tBHQ (12 μM), which mobilized the entire SERCA-sensitive ER calcium pool. (B) Ca²⁺ release measured in HL-60 cells treated with various concentrations of tBHQ (1-12 μM). (C) Ca²⁺ influx measured in HL-60 cells treated with various concentrations of tBHQ (1-12 μM). Mean ± SEM (n = 3).