Cellevent caspase 3 7 green flow cytometry assay kit

Mock- and ZIKV-infected (MOI 5) SH-SY5Y cells (6 × 10⁴) grown in 48-well plates were harvested at the indicated times post-infection, washed with PBS (phosphate buffered saline), and processed for FCM analysis. For cell death quantification, cells were stained with Annexin V (FITC) and 7-Aminoactinomycin D (7-AAD), according to the manufacturer’s instructions (BD Pharmingen, San Jose, CA, USA). For caspase-3/7 and SYTOX staining, cells were processed using a CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (Molecular Probes, Invitrogen, OR, USA) according to the manufacturer’s instructions. For cleaved PARP labeling, cells were labeled with mouse anti-human cleaved PARP (1:20, #552933, BD Biosciences, Franklin Lakes, NJ, USA) and 7-AAD, according to the manufacturer’s instructions. All FCM analysis was performed using BD FACSAriaIII Cell Sorter (BD Biosciences, Franklin Lakes, NJ, USA) and data analysis was performed using BD FACSDiva (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo (TreeStar, Woodburn, OR, USA) software.

HUVECs were collected and counted. Cell were stained with CellEvent Caspase-3/7 green flow cytometry assay kit (Molecular Probes) according to manufacturer's instruction. Cells were then analysed by FACS (BD LSR II or FACSARIA III system; BD Biosciences). Cells were gated and analysed by FlowJo software. In separate experiments, HUVECs were harvested at 48 h and labelled with Image-iT DEAD Green viability stain (Molecular Probes) and subjected to FACS analysis as above.

Obatoclax mesylate (OBAT) was purchased from Selleck Chemicals (Munich, Germany). The OBAT was dissolved in DMSO and stored as 5 mM stock solutions at −20 °C. RPMI 1640 medium and fetal calf serum were obtained from GIBCO BRL Life Technologies (Gaithersburg, MD, USA). L-glutamine, antibiotic antimycotic solution (AAS), dimethyl sulfoxide (DMSO), NBT, phorbol 12-myristate 13-acetate (PMA) and anti-β-actin antibody (cat. #A5316) were purchased from Sigma Aldrich (St. Louis, MO, USA). The FITC Annexin V Apoptosis Detection Kit I, propidium iodide (PI)/RNase staining buffer and anti-CD-11b were obtained from BD Biosciences (San Jose, CA, USA). The CellEvent™ Caspase 3/7 Green Flow Cytometry Assay kit was purchased from Molecular Probes (Eugene, OR, USA). The Hemacolor® Rapid Staining kit was obtained from Merck Millipore (Darmstadt, Germany). The antibody against Bcl-2 (cat. #2876) and horseradish peroxidase-conjugated secondary antibody (cat. #7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

To evaluate the effect of 5,6 α/β-ECs on caspase 3/7 activity, the cleavage of the Ac-DEVD substrate was measured using the CellEvent caspase 3/7 green flow cytometry assay kit (C10740, Molecular Probes, Eugene, OR, USA) [20 (link)]. JJN3 and U266 cells were seeded at a density of 2 × 10⁵ cells/mL/well in 24-well culture plates for 24 h and treated with 20–80 µg/mL of 5,6 α/β-ECs or with 500 μM H₂O₂ as positive control for 24–72 h. Next, cells were processed according to the manufacturer instructions. At least 10⁴ cells per sample were acquired with BD FACSCanto II and data were analyzed with the BD FACSDiva 7 software (BD Biosciences, Franklin Lakes, NJ, USA).

TASK-1 siRNA or control siRNA transfected cells were replated at 2x10⁴ cells/cm². After 24 hours apoptotic stimuli were added: either cisplatin, or DMEM medium lacking glucose (Gibco). After additional 72 hours floating cells and attached cells were harvested and the suspension was centrifuged at 400 g for 5 min. The percentage of apoptotic cells was determined with the Caspase-3 Intracellular Activity Assay Kit I (PhiPhiLux^® G1D2, Merck, Darmstadt, Germany) or, after discontinuation of the kit by the manufacturer, by the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Molecular Probes, Waltham, MA). The DEVD peptide concentration was set to 4 μM. Samples were analyzed by flow cytometry (FACS Calibur, BD Biosciences, San Jose, USA). As a second method cells were harvested, centrifuged, stained with Hoechst dye (Invitrogen, Waltham, MA), and nuclear fragmentation was assessed. The observer (KL) was blinded to the treatment, at least 500 cells per sample were evaluated.