Annexin 5 af647

After incubation with ALA alone or ALA plus light, the cells were centrifuged and the supernatants were kept at −80 °C for the isolation, labelling, and measurements of cytokines and exosomes as described in Section 2.7. The cells were then incubated in PBS (2% FBS) with an antibody concentration of 2 µL/mL and AnnexinV concentration of 50 µL/mL. The cells were washed with PBS (2% FBS), centrifuged, and the supernatants removed. The amounts of ALA-induced PpIX (1 h antibody incubation) and cell viabilities (1.5 h antibody incubation) in the individual subsets of PBMCs were measured with 4 antibody/dye combination methods: (1) CD3-FITC (Invitrogen, Carlbad, CA, USA), CD19-PE (ImmunoTools GmbH, Friesoythe, Germany), Fixable viability dye-eF450 (Invitrogen), AnnexinV-AF647 (Life Technologies, Eugene, OR, USA); (2) CD3-FITC, CD56-APC-eF780 (Invitrogen), Fixable viability dye-eF450, AnnexinV-PE (Invitrogen); (3) CD4-FITC (eBioscience, San Diego, CA, USA), CD8-PE (Invitrogen), Fixable viability dye-eF450, AnnexinV-AF647; (4) CD11c-FITC (ImmunoTools), CD14-PE/Cy7 (Invitrogen), Fixable viability dye-eF450, Annexin V-AF647. The measurements were done using a Cytoflex S cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA) with the Cytexpert software (Version 2.1, Beckman Coulter); and the analyses were performed using FlowJo software (Version 10, Treestar, Ashland, OR, USA).

PBMC were resuspended in RPMI 1640 plus 10% fetal calf serum at a concentration of 0.5 × 10⁶ cells/mL per well. Cells were incubated at 37 °C, 5% CO₂, for 20 h for the evaluation of spontaneous apoptosis. Apoptosis was detected by annexinV-AF647 (Invitrogen, San Jose, CA, USA) and propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA). PBMC were analyzed by flow cytometry after incubation with annexinV-AF647 for 15 min at RT and the addition of PI solution (2 μg/mL). AnnexinV-positive, PI-negative cells were considered to be undergoing apoptosis.