Luminescence imaging of ATP release from RBCs. (A) Image a is an IR-DIC image of RBCs before hypotonic shock application. Image b was obtained by subtracting control image a (before hypotonic shock) from that acquired ∼25 minutes after 20% hypotonic shock. The resulting white dots correspond to cells that lysed and were missing in images captured after hypotonic stimulation. c is an overlay of image b and cumulative ATP-dependent luminescence (in red) observed during the entire experiment. Note that regions of ATP release coincide with spots where cells lysed. (B) (a) Time-course of luminescence responses due to lysis of single RBCs in the experiment shown in A. Luminescence responses were measured at individual release sites seen in A and are indicated by traces of different colors. See supplemental Movie 1 for the entire time course of ATP release. (b) ATP release durations for the responses depicted in a measured from the start of release until the luminescence response decayed to 1/e of its peak value (black circles). Average (±SEM) release time was 17.6 ± 2.1 seconds (white square). (C) (a) Two examples (upper and lower row images) illustrating ATP release due to lysis of single RBCs. IR images of RBCs (in green) are overlaid with extracellular ATP-dependent luminescence (in red). 20% hypotonic shock-induced ATP release (center image) occurs shortly before lysis/collapse of a single RBC (indicated by a white arrow; see also supplemental Movie 2). No ATP release from intact RBCs is evident. The elapsed time (minutes:seconds) is shown in the upper-right corner. (b) Average delay time between start of ATP release and cell lysis for the responses shown in B (black circles). Average (±SEM) delay time was 47 ± 10.6 seconds (white square).