Figure C. No systematic differences in resting membrane potential and depolarisation in SCID T cells as compared to normal controls.

For measurements of the membrane potential, 1x10⁶ T cells were loaded with 1 mM of the coumarin lipid dye CC₂-DMPE (Aurora Biosciences, San Diego, CA) for 30 min at room temperature in 1 ml of serum free RPMI 1640 in the presence of 2 ml 10% w/v Pluronic-127/DMSO solution (Aurora Biosciences). The oxonol dye DiSBAC₂(3) (Aurora Biosciences) was loaded at 6 mM final concentration for 45 min at room temperature in RPMI 1640. Cells were loaded onto poly-L-lysine coated coverslips and analyzed by video imaging at 405 nm (excitation) and 460 and 570 nm (emission), respectively. Depolarisation was achieved by perfusing the cells in Ringer solution containing 20, 40, 60, 80, 100 and 155 mM KCl, respectively, with subsequent hyperpolarisation using standard 5 mM KCl Ringer solution. We show two of a series of paired experiments in which control and patient T cells were loaded simultaneously with CC₂-DMPE and DiSBAC₂(3) and directly compared. Results varied from experiment to experiment as a function time (i.e. how long the cells have been left loaded before starting the experiment). Taken together, however, we observed no systematic differences in membrane potential of SCID and control T cells.