Fcs express 7 research edition

Treated and untreated cells were stained for viability using blue fluorescent reactive dye (Invitrogen). Cells were washed and blocked using human BD Fc block (BD Biosciences) in 3% BSA in PBS and stained with anti-CD11c-BV421 (clone: Bu15; Biolegend). In-house generated rabbit anti-human PAD4 antibody was fluorescently labelled using AF647 antibody labelling kit (Invitrogen) and used at 0.01 µg µl⁻¹. Equal amounts of AF647-labelled rabbit IgG (3452S, Cell Signaling Technology) served as isotype control. Cells were incubated with labelled antibodies for 30 min at 4°C and flow cytometry was conducted at the Johns Hopkins Bayview Immunomics Core Facility using Cytek Aurora (Cytek Biosciences). Data were analysed using FCS Express 7 Research Edition (De Novo Software).

ADCP assays were performed using a reported protocol (30 (link)) with some modifications. FluoSpheres carboxylate-modified microspheres (Thermo Fisher, #F8823) were coupled with SARS-CoV-2 spike protein using the xMAP Antibody Coupling Kit (5 µg protein/~36.4x10⁹ beads, Luminex #40-50016). Spike-conjugated microspheres were incubated with diluted plasma for 2 hours at 37°C in the dark. After washing and centrifugation (2,000 g, 10 minutes), the beads (~3x10⁸ beads, 10 µL/well) were incubated with THP-1 cells (0.25x10⁵ cells, 200 µL/well) for 16 hours. The samples were analyzed on an Attune NxT flow cytometer (Thermo Fisher, #A24858). Data analysis was performed using FCS Express 7 Research Edition (De Novo Software).

To determine the cell cycle effects of the ATR, CHK1 and WEE1 inhibitors exponentially growing cells were exposed to DMSO, 3 µM cisplatin or 3 µM cisplatin plus either 1 μM VE-821, 50 nM PF-477736 or 100 nM MK-1775 for 24 h. Cells were washed twice with PBS, with each washing collected to ensure no loss of cells, before being trypsinised and harvested. Following centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the remaining cell pellet was resuspended in 1 mL ice cold PBS and centrifuged (3000 rpm, 5 min). The supernatant was removed, and 1 mL of 70% ethanol was added dropwise to the cell pellet. Samples remained at 4 °C for a minimum of 1 h. Prior to staining, cells were washed twice in PBS to remove ethanol before eventually being resuspended in 800 μL PBS. RNase was added to cells (final concentration 1 mg/mL) alongside propidium iodide (PI) stain (final concentration 400 μg/mL). Cells were incubated in dark conditions at 37 °C for a minimum of 30 min. Cells were analysed for DNA content using a BD FACSCanto II flow cytometer. Data were stored and transferred to FCS Express 7 research edition^®, De Novo software, Pasadena, CA 91107, USA for analysis. Doublets were excluded, and the sub-G1, G0/G1, S and G2/M populations were determined from the cell cycle histogram.