Lsr fortressa

The disturbance of the cell membrane was monitored using Annexin V (AnV) and propidium iodide (PI). Neutrophils (1 × 10⁶ cells/sample) were placed in an eppendorf tube, washed twice with PBS, and resuspended in solutions of FOH or FA at variable concentrations. Unstimulated cells served as a negative control, and phorbol 12-myristate 13-acetate (PMA)-treated neutrophils represented a positive control. Cells were incubated for 1 h at 37 °C, at 5% CO₂, washed three times with PBS, and stained with propidium iodide and fluorescein isothiocyanate (FITC)-labeled Annexin V for 15 min, according to supplier’s instruction (Dead Cell Apoptosis Kit with Annexin V-FITC and PI, Invitrogen, Carlsbad, CA, USA). Then, cells were analyzed with flow cytometry (LSR Fortressa, BD, San Jose, CA, USA).

To quantify apoptotic and necrotic cells, flow cytometry was performed. HaCaT cells (1 × 10⁶ cells/sample) were washed twice with cold PBS immediately after irradiation and centrifuged at 1000× g for 5 min. Pellets were suspended in annexin binding buffer and cells were incubated with FITC annexin V and PI for 15 min in room temperature. Next, 10⁴ unfixed cells per sample was analyzed with flow cytometry (LSR Fortressa, BD, San Jose, CA, USA) as described in detail elsewhere [86 (link)]. Three independent experiments were performed.

Neutrophils stimulated by selected factors were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC)-labeled Annexin V (AnV) (Dead Cell Apoptosis Kit with Annexin V-FITC and PI, Invitrogen, Carlsbad, CA, USA), according to the supplier’s instruction. The analysis of apoptosis progress was performed using flow cytometry (LSR Fortressa, BD, San Jose, CA, USA). In the second way, neutrophils were stained with the CellEvent™ Caspase-3/7 Detection Reagents kit (Invitrogen, Waltham, MA). The fluorescence was measured on a microplate fluorescence reader (H1, Biotek) - excitation: 495 nm, emission: 525 nm.

Cytokine-dependent cell lines were generated from transduced sorted bone marrow cells or from the cKit⁺ fraction of primary MN1-induced leukemic bone marrow after sorting and cultured in Dulbecco’s Modified Eagle Medium supplemented with 15% fetal bovine serum, 10 ng ml⁻¹ human IL6 (hIL6), 6 ng ml⁻¹ murine IL3 (mIL3) and 100 ng ml⁻¹ murine stem cell factor. For in vitro growth and proliferation assays, cells were sorted in triplicate 3 days after shRNA transduction using the BD FACSAria or BD FACSAria Fusion (both from BD Biosciences, San Diego, CA, USA) and counted using the Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Fullerton, CA, USA). For in vitro competitive assays, equal numbers of shRNA-transduced cells and untransduced MN1 cells were sorted by fluorescence-activated cell sorting, and the proportion of meKO2⁺ cells was analysed using the fluorescence-activated cell sorting LSRFortressa (BD Biosciences, San Jose, CA, USA).

Immediately after the isolation process, viability analysis of isolated neutrophils was performed, which was based on staining cells with propidium iodide (PI) and fluorescein isothiocyanate (FITC)-labeled Annexin V (AnV) (Dead Cell Apoptosis Kit with Annexin V-FITC and PI, Invitrogen, Carlsbad, CA, USA). Cells (1x10⁶) were washed three times with PBS and stained with PI and AnV for 15 minutes, according to the supplier’s instruction. Cell viability assessment was performed using flow cytometry (LSR Fortressa, BD, San Jose, CA, USA).