Cellular responses after exposure to rtPA were investigated to identify “early” apoptotic cells and to discriminate from necrotic/“late” apoptotic and vital cells. For simultaneous detection of apoptotic and necrotic cell death, a costaining technique with fluorochrome-conjugated Annexin V (Merck Biosciences, Bad Soden, Germany), in tandem with the DNA-binding dye propidium iodide (PI), was used according to the method of Vermes et al.
17 Externalization of phosphatidylserine occurs in the earlier stages of apoptosis, and Annexin V-FITC staining precedes the loss of membrane integrity that accompanies the latest stages of cell death (PI labeled), thus permitting the discrimination of early apoptotic cells from necrotic/late apoptotic cells.
18 Briefly, HCECs were grown to confluence and incubated for 24 hours in 24-well plates using the same conditions as in the previous sets of experiments. After centrifugation and washing in cold PBS, HCECs for Annexin V-FITC and PI staining were resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl
2, 2.5 mM CaCl
2, pH 7.4) at a concentration of 106 cells/mL. Five hundred microliters, containing 5 × 10
5 cells, was transferred to a culture tube, and 1.25 μL FITC-conjugated Annexin V was added. After centrifugation at 1000 rpm for 5 minutes and removal of the supernatant, cells were gently resuspended in 500 μL cold binding buffer, and 10 μL PI was added. Positive controls were provided for both apoptotic and necrotic (10% ethanol) cell death. For simultaneous scoring of the differential cellular response, aliquots of 10
4 cells each were immediately processed for fluorescence-activated cell sorting (FACS) on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA). Excitation parameters were set at λ
Ex = 488 nm, and fluorescence emission was detected at λ
Em = 518 nm for Annexin V-FITC and λ
Em = 620 nm for PI. Data analysis was performed with appropriate software (CellQuest; BD Biosciences, Mountain View, CA).