Annexin 5 fitc antibody

Nalm6 cells were grown in 6-well plates with a density of 80,000/mL and assay was performed as described [59 (link),60 (link),70 (link)]. Then the cells were treated with different doses of SP11 (0,0.4 μM, 0.8 μM, 1 μM) for 48 h. Cells were trypsinized, washed with ice-cold 1X Phosphate buffer saline and resuspended in 1X annexin binding buffer containing annexin V-FITC antibody (Biolegend, San Diego, CA, USA) for 15 min in the dark on ice. PI (propidium iodide) was added (3.3 μg/mL) just before acquiring the samples. In total, 10,000 events were acquired for each sample using Beckman coulter Gallios flow cytometer (Beckman Coulter, Miami, FL, USA).

Recombinant purified TNFα, the annexin V-FITC antibody and the 7-AAD antibody were purchased from BioLegend (London, United Kingdom). Necrostatin-1_s (Nec-1_s) was obtained from BioVision, Inc. (Milpitas, CA, USA). The zVAD-fmk (herein referred to as zVAD) was purchased from Bachem (Weil, Germany). Erastin (era), GSK’872, Necrosulfonamide (NSA) and the anti-MLKL antibody (clone 3H1) were obtained from Calbiochem (Merck Millipore, Darmstadt, Germany). Synthesis of the ferrostatin derivative 16-86 and the ferroptosis inducer RSL3 were described previously [12 (link), 14 (link)]. Ferrostatin-1 (Fer-1) was purchased from Xcess Biosciences Inc. (San Diego, CA, USA). Dabrafenib was purchased from Selleckchem (Absource Diagnostics, Munich, Germany). GW806742X was purchased from Biomol (Hamburg, Germany). The monoclonal anti-ACSL4, anti-GPX4, the anti-human and anti-mouse monoclonal phospho-MLKL antibodies were all obtained from Abcam (Cambridge, United Kingdom). The β-actin antibody was purchased from Cell Signaling (Frankfurt, Germany).

To evaluate erythroid differentiation, cells were incubated for 30 minutes on ice with anti-CD36-PE (clone 5-271; BioLegend), anti-CD71-FITC (clone OKT9; eBioscience), and anti–CD235a-APC (clone HIR2; BioLegend) antibodies before staining with 0.5 μg/ml DAPI (Sigma-Aldrich). To evaluate granulomonocytic differentiation, cells were first incubated for 30 minutes on ice with Fixable Viability Dye eFluor 780 (eBioscience). After washing with PBS, cells were incubated on ice for 30 minutes with anti-CD11b Brilliant Violet 421 (clone ICRF44; BioLegend), anti–CD14-FITC (clone RMO52; Beckman Coulter), and anti–CD15-APC (clone W6D3; BioLegend) antibodies. To perform apoptosis assays, cells resuspended in annexin V–binding buffer (BioLegend) were stained with annexin V-FITC antibody (BioLegend) for 20 minutes in the dark according to the manufacturer’s instructions. Propidium iodide (1 μg/ml) was added to the cells before analysis. To perform cell-cycle analysis, cells were fixed with ice-cold absolute ethanol for 30 minutes before incubation with 40 μg/ml propidium iodide and 10 μg/ml RNase A for 1 hour at 37°C. Flow cytometry was performed on a BD LSRII (BD Biosciences), and the data were analyzed using FlowJo software, version 7.6.4.