Calcium green

Cells were labeled with the fluorescent calcium indicator (Calcium Green, Invitrogen) by washing with HBSS and then adding 5 μM of fluorophore diluted in 2ml of HBSS. The cell-dye solution was incubated at 37°C for 45 minutes and then washed twice with HBSS to remove the excess fluorophore. This procedure results in almost complete cell labeling. To determined changes in calcium levels with stimulation and/or stretch, we imaged the calls as described below, and monitored the change in intensity of individual cells as a function of time during the study. The images were transferred to ImageJ to select the region of each cell (i.e. ROI) and the change in intensity over the time series was calculated. The data shown in the figures are compiled over many cells in several independent experiments were the fluorescence intensities were normalized to the initial point, and are presented in arbitrary units. To view cell behavior in the absence of extracellular calcium, A10 cells labeled with Calcium Green (Invitrogen) and HBSS containing 0.63 mM filtered EDTA was added immediately before stretching.

For measurement of intracellular calcium, non-adherent and adherent WT and IkE5^Δ/Δ pre-B cells were stained with Fura-red (Life technologies) as per manufacturer's protocol. For calcium flux, cells were harvested into staining buffer that contained 25mM Hepes (pH 7.2), 125 mM NaCl, 5mMKCl, 1mM Na₂HPO4, 0.1% glucose and 0.5mM MgCl_2, 1 mMCaCl₂ and 0.1g BSA just prior to use. Calcium green (Life technologies) and Fura-red were added for 30 minutes at 37°C. Cells were washed twice and re-suspended in staining buffer and placed on ice. Just prior to analysis on FACSCanto™ (BD), the cells were equilibrated to 37°C. Data was acquired for 30 seconds and then pulsed with anti-IgM antibody or ionomycin and acquired for additional indicated time points. Data was analyzed using kinetics platform on FlowJo software (Tree Star).