Anti cd61

Indirect immunofluorescence was used to detect the expression of differentiation- associated antigens on the surface of leukemic cells. Cells on days 1, 3, and 5 were washed with phosphate buffered saline (PBS) and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (goat F(ab’)2 anti-mouse IgG, (Cappel Laboratories, Cochranville, PA, USA) with primary monoclonal antibodies. Differentiated antigens were used to detect differentiation. Examples included anti-CD14 (BD Biosciences) for monocytes [50 (link)], anti-CD16 [51 (link)] (Miltenyi Biotec, Bergisch Gladbach, Germany) for neutrophils, anti-CD235a (BD Biosciences) for erythrocytes, and anti-CD61 [52 (link)] (BD Biosciences) for megakaryocytes. The background threshold was obtained using a FITC-conjugated goat anti-mouse IgG antibody. Flow cytometry was conducted using a FACSCalibur with an Argon-ion laser and a 488 nm emission filter, and the data were analyzed using the CellQuest^Pro software 5.1 (Becton Dickinson).

Cells were seeded gently onto glass coverslips coated with 0.001% poly-L-lysine in a 24-well plate in triplicate, allowed to settle, and incubated for 6 h at 37°C, 5% CO₂. After the incubation period, samples were gently fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, washed, and blocked with 3% BSA for 1 h. DNA was stained with propidium iodide (Vector Laboratories, Burlingame, CA, USA) and elastase using a specific anti-human neutrophil elastase antibody (Merk Millipore, Darmstadt, Germany). Plts were stained using anti-CD61 (BD Biosciences, San José, CA, USA), or the corresponding Ig isotype controls were used for setting non-specific binding (BD Biosciences, San José, CA, USA). Images for NET evaluation were acquired using a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan) equipped with a Plapon 60×/1.42 objective lens and processed using Olympus. At least 10 different fields were observed in each triplicate (200×). NET areas were determined as previously reported in at least five pictures obtained in 200× (Rodriguez-Rodrigues et al., 2017 (link)), using the wand tool from the FIJI software (Schindelin et al., 2012 (link)). The scale for the measurement was obtained from the data given in the confocal microscope image.

An indirect immunofluorescence method was used to detect the expression of differentiation-associated antigens on the surface of leukemic cells. Cells collected from day-5 cultures were cultured with primary monoclonal antibodies, washed with PBS, and exposed to FITC-conjugated secondary antibodies, i.e., goat F(ab’)₂ anti-mouse IgG (Cappel Laboratories, Cochranville, PA, USA). Monoclonal antibodies used included anti-CD14 (BD Biosciences) for monocyte, anti-CD16 (Miltenyi Biotec, Bergisch Gladbach, Germany) for neutrophil, anti-CD235a (BD Biosciences) for erythrocyte, and anti-CD61 (BD Biosciences) for megakaryocyte. FITC conjugated to goat anti-mouse IgG was used to set background thresholds. Flow cytometry with FACScaliber and data analysis using CellQuest ^Pro software (Becton Dickinson) were carried out.