Thp 1 monocytic cells

To establish an inflammatory model, THP-1 monocytic cells (ATCC, USA) were seeded into cell culture flasks. By adding 100 nM phorbol-12-myristate 13-acetate (Sigma-Aldrich, Germany), THP-1 monocytic cells were differentiated into macrophages for 3 days. Then, the PMA-containing medium was removed. The differentiation of PMA-treated THP-1 cells was extended by incubating the cells in fresh culture medium for an additional 5 days. Cells that continued to receive regular growth medium were designated as nonactivated macrophages (NM).

THP-1 monocytic cells was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). RPMI medium, glutamine, and antibiotics (penicillin and streptomycin), phosphate buffered saline (PBS), phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma–Aldrich (Poznań, Poland). Fetal bovine serum was purchased from Gibco (Gibco, Paisley, UK). Prostaglandin E₂ EIA Kit and Thromboxane B₂ EIA Kit were purchased from Cayman Chemical (Ann Arbor, MI, USA); Micro BCA Protein Assay kit was purchased from Thermo Scientific (Pierce Biotechnology, Rockford, IL, USA).

All cells were cultured at 37 °C and 5% CO₂. THP-1 monocytic cells (American Type Culture Collection, Manassas, VA, #TIB-202) were cultured in Roswell Park Memorial Institute 1640 Media (RPMI 1640) (Gibco, Grand Island, NY, #118875-093) supplemented with 10% v/v fetal bovine serum (FBS) (GE Cytiva, Marlborough, MA #SH30541.03), 100 U/mL penicillin-streptomycin (Gibco #15140-122), 10mM HEPES (Gibco #15630-080), 1mM sodium pyruvate (Gibco #11360-070), 2 g/L D-Glucose (Gibco #A24940-01), and 1X GlutaMAX supplement (Gibco #35050-061). HEK 293T/17 cells (American Type Culture Collection #CRL-11268) and HeLa cells (American Type Culture Collection #CCL-2) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco #11965-092) supplemented with 10% fetal bovine serum, and 100 U/mL penicillin-streptomycin.

THP-1 monocytic cells (American Type Culture Collection) were inoculated with EBOV/BDBV-GP virus expressing eGFP at MOI of 2 PFU/cell, incubated for 48 hours, washed two times to remove unbound virus, and incubated with 100 μg/ml of mAbs or no mAb. Following a one hour-long incubation, cells were placed atop of monolayers of Vero-E6 cells pre-stained with CellTrace Far Red (ThermoFisher Scientific) according to the manufacturer’s recommendations, incubated for 72 hours and fixed with 4% paraformaldehyde. Cells were analyzed by flow cytometry to determine the percentages of cells double-positive for CellTrace Far Red and eGFP of total cells positive for CellTrace Far Red. The percentage of double-positive cells indicated the percentage of cells that became infected due to cell-to-cell transmission of virus. For each sample, 30,000 events were counted. In a separate experiment, supernatant aliquots were harvested from co-cultures of THP-1 and Vero-E6 cells on days 3–5 after the inoculation of monocytes and then titrated on Vero-E6 cell monolayers.

THP-1 monocytic cells (American Type Culture Collection, Manassas, VA, USA) growing in continuous culture were differentiated into macrophages following phorbol myristate acetate (PMA) stimulation for 72 h as previously reported [4] (link), and cells treated as described for alveolar macrophages.

ADCP was measured using monocytic THP-1 cells (ATCC, Manassas, VA), as previously described (42 (link)). Briefly, CRF01_AE gp140 protein was biotinylated at a biotin to gp140 ratio of 50 following manufacturer’s instructions (Thermo Fisher, Waltham, MA) and excess biotin was removed using Zeba desalting columns (Thermo Fisher, Waltham, MA). Biotinylated gp140 was incubated with yellow-green streptavidin-fluorescent beads (Molecular Probes, Eugene, OR) for 2 hours at 37°C. The gp140 beads were diluted 100-fold. In a 96-well plate, 10μl of diluted beads were mixed with diluted mother (1:3000) or infant (1:1000) plasma then incubated 2h at 37°C. Healthy donor plasma was utilized as a negative control. THP-1 cells (20,000 cells per well) were added and plates were incubated at 37°C for 18 hours. Cells were fixed with 4% paraformaldehyde solution and fluorescence was evaluated on an LSRII flow cytometer (BD Bioscience, Franklin Lakes, NJ). The phagocytic score was calculated by multiplying the percentage of bead-positive cells by the geometric mean fluorescence intensity of the bead-positive cells and dividing by 10⁴.