Heat inactivated fbs

The human biological samples were sourced ethically following GSK-HBSM guideline and policies, their research use was in accord with the terms of the informed consents. THP-1 cells (human monocytic leukemia) were made available by GSK-Biological Reagents and Assay Development Department (GSK-BRAD, Stevenage, UK) and were maintained in RPMI media (Life-Technologies) supplemented with 1.25 mM Pyruvate (Life-Technologies), 2.5 mM Glutamine (Life-Technologies), 25 mM HEPES (Life-Technologies) and 10% heat inactivated FBS (Gibco).
The Leishmania donovani strain (MHOM/SD/62/1S-CL2D, LdBOB) [20 (link)] expressing green fluorescent protein (GFP) was kindly provided by Manu de Rycker (University of Dundee) [21 (link)]. Axenic amastigotes were grown at 37°C, 5% CO₂ in media containing 15 mM KCl (Invitrogen^™), 10 mM K₂HPO₄ (Merck), 136 mM KH₂PO₄ (Merck), 0.5 mM MgSO₄ (Sigma-Aldrich), 24 mM NaHCO₃ (Invitrogen^™), 25 mM Glucose (Sigma-Aldrich), 1mM L-Glutamine (Invitrogen^™), 1xRPMI Vitamin Solution (Sigma-Aldrich), 10 μM Folic Acid (Sigma-Aldrich), 100 μM Adenosine (Sigma-Aldrich), 5 mg/L Hemin (Sigma-Aldrich), 1xRPMI Amino Acid solution (Sigma-Aldrich), 25 mM MES, 0.0004% Phenol Red and 20% heat inactivated FBS (Gibco) in Milli-Q water (pH = 5,5 at 37°C). The selection antibody Lexsy NTC (Nourseothricin, Jena Bioscience) was regularly added to amastigote cultures [21 (link)].

PAMs were obtained from lung lavage of 6–8-week-old specific pathogen-free (SPF) piglets (the Large White breed). PPMs were obtained from the peritoneal lavage of SPF pigs. Peripheral blood monocytes (PBMC) were isolated from SPF pigs by Ficoll-Paque (Sigma, Saint Louis, MO, USA) density gradient centrifugation according to the manufacturer’s instructions. PAMs, PPMs, and PBMC were cultured in RPMI 1640 (Gibco, Grand Island, NE,) with 10% heat-inactivated FBS (Gibco, Grand Island, NE, USA), 1% penicillin and streptomycin. Marc-145 cells were maintained in Dulbecco modified Eagle medium DMEM (Gibco, Grand Island, NE, USA) supplemented with 10% heat-inactivated FBS (Gibco, Grand Island, NE, USA), 1% penicillin and streptomycin. L929 cell culture supernatant was harvested as previously described [31 (link)]. All cells were maintained at 37 °C in an incubator with 5% CO₂.
The highly pathogenic PRRSV (HP-PRRSV, PRRSV-2) (GenBank accession, JX317648) was propagated and titrated in PAMs and Marc-145 cells. The porcine epidemic diarrhea virus (PEDV) strain was propagated and titrated in Marc-145 cells. The viral supernatants from cell cultures were collected at different time points after virus inoculation, and the determination of viral 50% tissue culture infective doses (TCID₅₀) was performed using the Reed–Muench method [32 (link)].

African green monkey (Cercopithecus aethiops) kidney epithelial cells (Vero E6 cells; ATCC CRL-1586) were maintained in complete DMEM containing 1× DMEM supplemented with 25 mM HEPES, 2 mM l-glutamine,1 mM sodium pyruvate, 1× non-essential amino acids, 1× antibiotic/antimycotic solution (all from Corning Life Sciences) and 10% heat-inactivated FBS (Life Technologies), unless indicated otherwise. Human lung adenocarcinoma epithelial cells (Calu-3 cells; ATCC HTB-55) were maintained in complete MEM (cMEM) containing 1× MEM (Corning Life Sciences) supplemented with 1× antibiotic/antimycotic solution and 10% heat-inactivated FBS unless indicated otherwise. Primary leukocytes from the airways of severe COVID-19 patients were collected bedside via endotracheal aspiration, and whole blood was collected by standard venipuncture, then processed as previously described (D.J. Eddins et al., manuscript posted on bioRxiv, DOI: 10.1101/2021.06.02.446468). SARS-CoV-2 USA-WA1/2020 (hereafter SCV2-WA1) was provided by BEI Resources (Manassas, VA, USA). Virus was propagated in Vero E6 cells as previously described (15 , 31 ), and titer was determined by 50% tissue culture infective dose (TCID₅₀/ml) or plaque assays (PFU/ml). Low-passage (P1 or P2) virus stocks were used throughout this study.

Breast cancer cell lines were purchased from the American Type Culture Collection (ATCC) and authenticated by DNA profiling using short tandem repeat (GenePrint^® 10 System, Promega) at Genomics Core Facility, Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM. MDA.MB.231 and MCF7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% heat-inactivated FBS (Life Technologies) and antibiotics (100 U mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin) (Life Technologies). Cells were maintained at 37 °C in a 5% CO₂ humidified incubator. The FBS used for exosome purification experiments was depleted of EVs by centrifugation at 100,000g for 1 h 10 min at 4 °C. For the preparation of medium with EV-depleted 0.5% FBS, EV-depleted FBS was added to DMEM to a final concentration of 0.5% v/v.

K562 cells and the cells stable transfected with the Kusabira-Orange (KOr) fluorescent protein or blue fluorescent protein (BFP) were, respectively, cultured in the basic RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (Life Technologies), 100 U penicillin G/ml, and 100 µg streptomycin/ml (Sigma-Aldrich) at 37°C in 5% CO₂. The cell proliferation assay was performed using the CellTiter-Glo luminescent cell viability assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.

Transendothelial migration (TEM) assays were carried out as previously described (22 (link)). Briefly, 6.5-μm Transwell inserts with 3-μm polycarbonate membranes (Corning Costar U.K.) were coated overnight with 2 μg/ml fibronectin and seeded with 5 × 10⁴ bEnd5 cells grown in DMEM supplemented with 10% heat-inactivated FBS (both Life Technologies) as described. Confluent bEnd5 cells were stimulated for 16 h with 5 nM TNF-α, and 5 × 10⁵ neutrophils were added into washed inserts that had been placed into wells of 24-well plates in the presence of 0, 1, or 3 nM MIP2 and allowed to migrate toward the chemoattractant. Transmigrated neutrophils were labeled for GR1, and 8 randomly chosen fields of view per 24 wells were photographed for counting (×20 magnification using an Evos cell imaging system [AMG]).