Annexin 5 fitc

HT-29 cells were grown in McCoys 5A media supplemented with 10% FBS, and seeded in 6-well plates at a density of 300,000 cells per well. The next day, isofagomine or noeuromycin (500 µM or 250 µM) was added and incubated for 30 min. Next, TcdA (1 nM) was added to each well and cells were incubated at 37 °C, 5% CO₂ for 24 h. Cells were harvested using 0.25% trypsin, filtered through 40 µm cell strainer caps into 5 mL polystyrene tubes and pelleted by centrifugation (1500 rpm, 5 min). Cells were stained for AnnexinV-FITC (Biolegend #640945) as per the manufacturer’s instructions. Briefly, cells were washed twice with staining buffer, then resuspended in 100 µL binding buffer. AnnexinV (5 µL) was added, and samples were incubated in the dark for 15 min at room temperature. Binding buffer (400 µL) was added and samples were analyzed on an LSRII flow cytometer (Becton Dickson). Data was analyzed using FlowJo (TreeStar) and Prism (Graphpad) software.

For proliferation assay, cells were detached by trypsinization, washed and left in PBS to a density of 10⁶cells/ml. CFSE (eBioscience) was added to a final concentration of 5 μM, mixed for 10 s and incubated for 10 min. One volume of FBS was added, followed by complete medium and cells were then pelleted, washed, seeded into p60 petri dishes, and incubated with DMSO (dimethyl sulfoxide) or silmitasertib for 48 h. For apoptosis assay, cells (2 × 10⁵) were washed with PBS and left in a total volume of 100 μl binding buffer (2.5 mM CaCl₂, 0.01 M Hepes, 0.14 M NaCl). One microliter annexin-V-FITC (Biolegend) and 2 μl 50 μg/ml propidium iodide (PI) were added and cells incubated for 15 min at RT. Finally, 300 μl binding buffer was added to stop the reaction. For cell cycle assay, cells were incubated with 25 μM silmitasertib for the indicated times and then centrifuged at 1000 x g for 5 min at RT, washed in 1 ml cold PBS, suspended in 1 ml staining solution containing 0.1% Triton X-100, 50 μg/ml PI and 200 μg/ml RNAse, and incubated for 30 min at 37 °C in the dark. All assays were analyzed on a Becton-Dickinson LSR Fortessa X-20 flow cytometer and the FACSDiva 8.02 software (San Jose, CA) at the MED.UCHILE-FACS Facility, Facultad de Medicina, Universidad de Chile.

The expression of Cleaved Caspase-3, as determined by Western blot, was used to evaluate podocyte apoptosis. Flow cytometry was also performed to assess the degree of apoptosis in podocytes. Annexin-V-FITC and PI (Bio Legend, USA) were used to identify apoptotic cells according to the manufacturer’s instructions. Cells in the upper-right and lower-right quadrants were classified as apoptotic.

Flow cytometry with dual staining of Annexin V and PI was performed as we previously described [26 (link)]. Briefly, following 24 h CSE exposure, cells were harvested by trypsinization. Approximately 10⁵ cells were stained in 1× binding buffer (0.01 M HEPES, pH 7.4; 0.14 M NaCl; 0.25 mM CaCl₂) using 5 μL of Annexin V-FITC (BioLegend, San Diego, CA, USA) and 10 μL PI (BioLegend). The cells were then incubated in the dark for 15 min at room temperature and the percentage of FITC- and PI-positive cells were quantified using FACS Canto-II flow cytometer (BD Biosciences, San Jose, CA, USA) and were analyzed using FlowJo software (version 7.6.3; TreeStar, San Carlos, CA, USA).

Anti-human CD66b, Annexin-V-Alexa647 and Annexin-V-FITC were purchased from BioLegend (San Diego, CA, USA). Calcein Blue AM was from Invitrogen (Waltham, MA, USA). Antibodies against MPO were from Dako (A0398). Ficoll-Paque Plus was from GE Biosciences (Baie d’Urfé, QC, Canada); endotoxin-free (<2 pg/mL) RPMI 1640 was from Wisent (St-Bruno, QC, Canada). Recombinant human cytokines were from R&D Systems (Minneapolis, MN, USA). N-formyl-methionyl-phenylalanine (fMLP) and phenylmethanesulphonyl fluoride (PMSF) were from Sigma (St. Louis, MO, USA). All inhibitors, antagonists, and fluorescent probes were purchased through Cedarlane Labs (Mississauga, ON, Canada). PlaNET reagents (fluorescent chromatin-binding polymers) are no longer available from Immune Biosolutions or other suppliers; we therefore employed a close equivalent: fluorescent, 50-nm carboxylate microspheres (# 16661-10) from Polysciences Inc. (Warrington, PA, USA). These microspheres are referred to as PlaNET reagents throughout this study, since the name is neither registered as a trademark, nor under copyright. All other reagents were of the highest available grade, and all buffers and solutions were prepared using pyrogen-free, clinical grade water.

Cells were plated into 35-mm plates 16–20 h prior to treatments. The following reagents were added to plates at the indicated concentrations and durations: Thapsigargin (3 μM) for 24 h, human recombinant TRAIL (25 ng/ml) for 5 h, ABT-737 (2.5 μM) for 16 h, UV (500 J/M²) for 16 h or UV/ABT-737 (500J/M²-2.5 μM), with ABT-737 (2.5 μM) added immediately following UV treatment for 16 h. Cells were also subjected to serum starvation by placing them in McCoy's 5A media without fetal bovine serum for 48 h. Cells were analyzed by either FACS or western blot against PARP following each treatment. For Annexin V analysis, treated cells were gathered from media in plates and trypsination of attached cells. Annexin V FITC (Biolegend, San Diego, CA, USA, #640906) was used to stain treated cells according to the manufacturer's instructions. Each sample was then analyzed for FITC signal by flow cytometry. A BD FACSCalibur flow cytometer with BD FACSDiva 8.0 software (UNMC, Flow Cytometry Research Facility) was used for cell counting and analysis. Detection of PARP cleavage in treated samples was achieved by preparing whole cell lysates as described above and subjecting lysate to western blot with PARP antibody.