Fig. 7. Effect of BAPTA and NAC on intracellular Ca²⁺ and ROS generation induced by partial irradiation. (A) Quantification of Fluo-3 fluorescence as a measure of intracellular Ca²⁺ levels in CMXRos-loaded cells (except for control cells) 30-60 minutes following partial irradiation. Prior to irradiation, CMXRos-loaded cells were either treated with BAPTA-AM (10 µM, 30 minutes) or NAC (15 mM, 30 minutes). Fluo-3 (2.5 µM) was subsequently loaded after irradiation and the fluorescence in partially irradiated cells was determined as described in Materials and Methods and expressed relative to non-irradiated cells in the same field (±standard deviation). Control cells for laser phototoxicity were neither stained with CMXRos nor treated with BAPTA and NAC prior to partial irradiation. The number of cells analysed in each group is indicated on top of each bar. Significant differences (*P<0.01; **P<0.001) between the group analysed in comparison with the group loaded with CMXRos but not treated with BAPTA or NAC were tested by student's unpaired t-test. (B) Representative images showing the increased Fluo-3 fluorescence (in this case 4.1-fold) in a partially irradiated cell compared with a non-irradiated cell. CMXRos was loaded prior to irradiation and Fluo-3 AM (2.5 µM) was loaded after irradiation. Region of irradiation is denoted by the dashed box and time after irradiation indicated in the top right. (C) Quantification of DCF fluorescence as a measure of intracellular ROS levels in CMXRos-loaded cells (except for control cells) 30-60 minutes following partial irradiation, as described in A. (D) Representative images showing increased DCF fluorescence in a partially irradiated cell (in this case 7.9-fold) collected similarly to those in B and detected by H₂DCFDA (40 µM) loaded after irradiation. This partially irradiated cell shows evidence of blebbing (45 minutes). Note that in B at 30 minutes and in D at 45 minutes the gain levels of CMXRos images were increased to permit visualisation of mitochondria in partially irradiated cells. In quantitative analyses of fluorescence care was taken to ensure that only adherent cells were imaged in all cases. Thus, the various cells depicted in B and D are imaged in the same focal plane in each case. The DCF fluorescence of non-irradiated cells in D is close to the background levels. Bars, 10 µm.