Fitc conjugated annexin 5

The apoptotic cells were identified by flow cytometry using the Annexin-V/FITC (BDBiosciences, San Diego, CA, USA) assay according to the manufacturer’s instructions. For detection of apoptosis and necrosis, FITC-labeled annexin-V combined with PI (propidium iodide) was used to mark the presence of phosphatidylserine (PS), which is displayed during apoptosis at the cell surface. PI only stains the nuclei of damaged cells with permeable plasma membranes. Briefly, the cells were incubated with tested compounds for 2 and 4 h. After incubation, the cells were washed twice with cold PBS and then resuspended in 100 μL of binding buffer, containing 2 μL of FITC conjugated annexin-V and 10 μg mL⁻¹ of PI (Becton-Dickinson, San Jose, CA, USA). Then, the preparations were incubated at room temperature, protected from light, for 15 min. Fluorescence was measured immediately after staining by flow cytometry using FL1 (green, annexin-V) and FL3 (red, PI) standard fluorescent filters.

Apoptotic or necrotic cell levels were determined by flow cytometry after double staining with Annexin V-FITC and propidium iodide using an assay kit from BD Pharmingen as described previously [17 (link)]. Briefly, 2 mL of cells (1 × 10⁶ cells/mL) were added to each well of 12 plates and treated with 5, 10, 20, 40 and 80 mg/mL of D-glucose for 2 h. Control well plates were also made without D-glucose. After 2 h of incubation, 1 × 10⁶ cells/mL were counted and washed in PBS, re-suspended in binding buffer (10 mm Hepes/NaOH pH 7·4, 140 mm NaCl, 2·5 mm CaCl₂), and stained with FITC-conjugated annexin V (Pharmingen, Becton Dickinson Co., San Diego, CA, USA). After staining, the cells were incubated for 15 min in the dark at room temperature. Cells were re-washed with binding buffer and analysed by flow cytometry (FACS Calibar; Becton-Dickinson) using Cell Quest software.

The following mAbs and reagents were used in this study: Allophycocyanin (APC)-Cy7-conjugated anti-CD3, phycoerythrin (PE)-Cy5-conjugated anti-CD161 and fluorescein isothiocyanate (FITC)-conjugated anti-TCR γδ, FITC-conjugated anti-CD3, FITC-conjugated anti-IFN-γ, FITC-conjugated annexin V, PE-conjugated anti-CD3, PE-conjugated anti-IL-17, PE-Cy7-conjugated anti-TNF-α, PE-conjugated anti-CD69, FITC-conjugated mouse IgG isotype, PE-conjugated mouse IgG isotype and PE-Cy7-conjugated mouse IgG isotype control (all from Becton Dickinson, San Diego, CA); PE-conjugated anti-programmed death-1 (anti-PD-1; eBioscience, San Diego, CA) and APC-conjugated anti-TCR Vα7.2 (BioLegend, San Diego, CA). Cells were stained with combinations of appropriate mAbs for 20 minutes at 4°C. Stained cells were analyzed on a Navios flow cytometer using Kaluza software (version 1.1; Beckman Coulter, Brea, CA).

Thymocytes were isolated following the procedure described previously^{3 (link)}. Irradiation was conducted about 1 h after cell culture in vitro. At 24 h after irradiation, thymocytes were collected, washed using PBS supplemented with Ca²⁺ and Mg²⁺, and then stained with a FITC-conjugated annexin V (BD Pharmigen) and propidium iodide (5 μg/ml, Sigma-Aldrich). Data were collected from at least 20,000 cells by FACSCanto (BD Pharmingen) and analyzed by Flowjo (Tree Star, Inc) without knowledge of the genotype or treatment by a single observer (C-LL).