Apoptosis detection kit

U251 cells treated with either C1 or DMSO for 48 hours was analyzed by flow cytometry with Annexin V antibody and Propidium Iodide (Life technologies, NY) using the Apoptosis Detection Kit (R&D Systems, MN) according to the manufacturer’s instructions. Data was confirmed by 3 independent experiments.

For apoptosis analysis, an Apoptosis Detection Kit was employed (4830-01-K; R&D Systems, Minneapolis, MN, USA). ARPE-19 cells were treated with Trypsin (15400054-Gibco^® Thermo Fisher Scientific, Dreieich, Germany) before being collected by centrifugation and washed twice in PBS (BE02-017F-Lonza, Walkersville, MD, USA). The suspension of cells was stained with a 1:100 dilution of the TACS Annexin V-FITC and a 1:10 dilution of Propidium lodide (PI) for 15 min in the dark; resuspended in the provided binding buffer and immediately processed by flow cytometry. Cr–Au was used as the negative control. A total of 10,000 events per sample of ARPE-19 cells were acquired with the flow cytometer (CytoFlex V2, Beckman Coulter, Inc., Brea, CA, USA). All the data were analyzed by CytExpert V.7.6 (Tree Star, Inc., Ashland, OR, USA) and Kaluza V 2.1 (Beckman Coulter, Inc., Brea, CA, USA). The gating strategy is showed in Supplementary Figure S2E.

Annexin-V staining was performed using an apoptosis detection kit (R&D Systems). Both adherent and non-adherent cells were collected, washed with PBS, and resuspended in binding buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl₂. After 15 min incubation of Annexin-V-FITC antibody at room temperature, cells were analyzed using a FACScan flow cytometer (Becton Dickinson).

Cell death was measured through the Annexin V-FITC/PI (fluorescein isothiocyanate/propidium iodide) Apoptosis Detection Kit (R&D Systems, USA), according to the direction from the manufacturer. The fluorescence intensity of Annexin V-FITC/PI was detected through flow cytometry (BD Bioscience).
For quantifying the renal macrophage polarization in mouse kidneys, kidney tissues were minced into 1 mm³ fragments and then digested in RPMI 1640 buffer containing 100 U/mL DNase I and 2 mg/mL collagenase type D for 60 min at 37°C and then passed through a 70 μm mesh to get single-cell suspension. Red blood cell lysis buffer (Sigma, USA) was used for lysing the red blood cells in the suspension. Mps were centrifuged and then resuspended in FACS buffer on ice. Incubated with 2.5 μg/mL Fc-blocking solution, Mps were resuspended in FACS buffer. Then, 10⁶ cells were stained with 3 fluorochrome-labeled antibodies: F4/80 (eBioscience)-PE, CD11c-FITC, and CD206-FITC. Finally, Mps were detected immediately on a FACS Canto II cytometer with DIVA software (Becton Dickinson). The data were analyzed by Cyflogic V.1.2.1 software.

Annexin V staining using an apoptosis detection kit (R&D Systems) was detected through flow cytometry according to the manufacturer’s instructions.

L. donovani promastigotes in the stationary phase (2×10⁷cells/ml) were seeded at 10⁶cells/ml in M199 medium and incubated for 48 hrs, at 26°C with the GI₅₀ concentration of each analogue or with DMSO, as control. For the analysis of the cell cycle, the procedure described in Georgopoulou et al.[28 (link)], was followed to prepare the samples for fluorescence-activated cell sorting. In order to determine the amount of cells, which display membrane fluidity perturbations, FACS analysis was performed using AnnexinV-FITC and propidium iodide (PI) staining (Apoptosis Detection kit, R & D Systems). All samples were analyzed using a Becton Dickinson FACS Calibur flow cytometer and data were analyzed using the Cell Quest software. All experiments were performed in triplicates.