Calcium measurements

Changes in intracellular concentrations of free calcium ([Ca²⁺]_i) were analyzed using fluo-3-AM (BD Pharmingen, Waltham, MA, USA), as previously described.⁵⁷ Neutrophils, at a concentration of 2.5 × 10⁶ cells/ml, were treated with 4 μM fluo-3-AM for 30 min at 30 °C. Then, cells were washed and resuspended at 5.0 × 10⁶ cells/ml in RPMI-1640 supplemented with 5% FCS. Aliquots of this cellular suspension (50 μl) were added to 400 μl ml of RPMI-1640 medium supplemented with 5% FCS, prewarmed at 37 or 39.5 °C. The prewarmed sample was immediately loaded onto the flow cytometer, and fluorescence was evaluated for ∼20 s. After this time, the medium was acidified, or not, by the addition of a predetermined volume of isotonic solution of HCl to adjust its pH to 6.0, and the fluorescence was recorded during an additional period of 100 s. A time-based gate was used to divide the original data file and separate cells according to the time at which their fluorescence in an FL1 detector was measured. The fluorescence of samples suspended at pH 7.3 and 37 °C were used to establish a marker at a FL1 fluorescence channel number greater than that exhibited by at least 97% of these resting cells. This marker was then used to determine the relative percentage of activated cells. Cells that raised their [Ca²⁺]_i to higher levels than that shown by 97% of resting cells in response to pH 6.0 and/or hyperthermia were considered to be activated.