Alexa fluor 488 conjugated annexin 5

Flow cytometry was performed using FACS Verse Cell Sorter (BD Biosciences) and analysed using FlowJo software. For Annexin-V staining, cells were labelled with Alexa Fluor 488-conjugated Annexin-V for 15 min in Annexin V-binding buffer according to the manufacturer’s instructions (Invitrogen). Click-it EdU kit (Invitrogen) was employed to assess the cell cycle profile and quantify the percentage of S-phase cells. Briefly, cells were incubated with EdU (5-ethynyl-2′-deoxyuridine) at a concentration of 10 μM for 20 min and subsequently, detection of EdU was performed according to manufacturer’s instructions (Invitrogen). To allow for quantification of mitotic cells and RRM2- or p-RPA/γH2AX- or PCNA-γH2AX-positive cells, cells were further labelled with anti-H3^Ser10, anti-RRM2, anti-p-RPA+anti-γH2AX or anti-PCNA+γH2AX antibodies, respectively. For G2/M arrest experiment, cells were treated with nocodazole (0.04 μg ml⁻¹, Sigma-Aldrich) for 12 h prior the analysis. In all analyses, DNA was counterstained with Hoechst 33342 dye (Invitrogen). Measurement of ROS (H2-DCF probe, Invitrogen) and 8-oxo-2′-deoxyguanosine (using anti-8-oxo-2′-deoxyguanosine antibody followed by Alexa Fluor 488-conjugated secondary antibody) levels were measured as described previously22 (link). Samples were acquired using FACSverse (BD Biosciences) and FlowJo software was used for data analysis.

Cells were seeded in 6-well culture plates and were induced to apoptosis by a treatment of tunicamycin for 48 h or gemcitabine for 72 h. The floating and adherent cells were collected, then co-stained with Alexa Fluor 488- conjugated Annexin V (Invitrogen) and PI. Samples were run within 30 min using flow cytometry. FlowJo software was used to analyze the data.

Over the 24 h period following UVB exposure cell viability was determined by dead cell apoptosis kit containing propidium iodide/Alexa Fluor 488-conjugated Annexin V (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Labelled cells were analyzed by flow cytometry with a FACSCalibur (Becton Dickinson, San Jose, CA, USA) measuring the fluorescence emission in FL1 (530 nm) and FL3 (>575 nm). Double negative cells represent viable cells. For data collection and evaluation CellQuest software 5.2 (Becton Dickinson) and Flowjo single cell analysis software were used.
Cell proliferation was determined by clonogenic assay. Cells were seeded in 100 mm Petri dish at 5000 cells/dish and were allowed to grow for 10 days. The medium was replaced each day to monitor the long-term effect of cell treatments. Ten days later, cells were washed with PBS, fixed with 100% methanol, and stained with May-Grünwald-Giemsa solution (Histolab Products, Västra Frölunda, Sweden).

Apoptotic cells were assessed using a cytometric assay kit (Invitrogen, Temecula, CA, USA) to confirm the protective effect of A. absinthium extract or shikimic acid against cisplatin-induced apoptosis [85 (link)]. LLC-PK1 cells were seeded in 6-well plates at a density of 1 × 10⁶ cells in 2 mL per well for 24 h (5% CO₂ and 37 °C). The cells were then treated with A. absinthium extract or shikimic acid at various concentrations with or without 25 µM cisplatin. DMSO was used as a control, and NAC was used as a positive control. After 24 h, the cells were harvested and stained with Alexa Fluor 488-conjugated annexin V (Invitrogen, Temecula, CA, USA) for 20 min in the dark, followed by washing with DPBS. Fluorescence images of the cells were captured using a Tali Image-based Cytometer (Invitrogen, Temecula, CA, USA). The number of apoptotic cells was determined using TaliPCApp software (version 1.0). The results were calculated as the percentage of cells stained with annexin V versus the total number of cells in each group.

To study the externalization of phosphatidylserine, cultures were placed in a glass-bottomed Perspex chamber in 0.5 ml HBHBSS and 5 μl of Alexa-Fluor 488-conjugated annexin V (Invitrogen) was added and mixed rapidly. Images were acquired with wide-field microscopy at fixed timepoints (usually at 5–8 and 10–13 minutes) after the addition of annexin V using a Zeiss Axioplan II microscope equipped with a 63× water immersion lens, an FITC filter set, and a 100 W AttoArc HBO mercury vapor lamp. Differential interference contrast images were captured just prior to the fluorescent images.