Fetal calf serum fcs

RPMI1640 medium, trypsin, Triton X-100, Brij 35, TriReagent and hexadimethrine bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nonidet P-40 was from Nacalai Tesque (Kyoto, Japan). Bovine serum albumin (BSA), gelatin, dimethyl sulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA) and polyethylene glycol (PEG) 6000 were purchased from Wako Pure Chemical Industries (Osaka, Japan). Polyethylenimine “Max” was purchased from Polysciences Inc. (Warrington, PA, USA). Fetal calf serum (FCS) was obtained from Biosera (Boussens, France). ASF104 serum-free medium was supplied by Ajinomoto (Tokyo, Japan). Matrigel was purchased from BD Biosciences (San Diego, CA, USA). The PrimeScript RT Reagent Kit and KAPA SYBR FAST qPCR Kit Master Mix (2×) ABI Prism were obtained from Takara Bio Inc. (Shiga, Japan) and KAPA Biosystems (Boston, MA, USA), respectively. ViraPower Lentiviral Packaging Mix and puromycin dihydrochloride were supplied by Life Technologies (Carlsbad, CA, USA). Oligonucleotides were supplied by FASMAC (Kanagawa, Japan). Recombinant murine TNF-α was a product of Peprotech (Rocky Hill, NJ, USA). A fluorescent dye, 3′-O-acetyl-2′, 7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein, diacetoxymethyl ester (BCECF-AM), was obtained from Dojindo Laboratories (Kumamoto, Japan). Gelatin Sepharose 4B was a product of GE Healthcare (Piscataway, NJ, USA).

The immortalized normal skin-derived human keratinocyte line N/TERT was maintained in keratinocyte serum-free medium (KSFM) (ThermoFisher Scientific, Waltham, MA, USA) as described previously [5 (link),23 (link)]. T8 cutaneous squamous cell carcinoma cell line (gift from Prof. Catherine Harwood) was cultured in a complete keratinocyte growth medium (KGM containing Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Basel, Switzerland): Ham’s F-12 (ThermoFisher Scientific, Waltham, MA, USA) in the ratio of 3:1 supplemented with 10% fetal calf serum (FCS) (Biosera, San Diego, CA, USA), epidermal growth factor (EGF) (Invitrogen, Waltham, MA, USA), insulin human solution (Sigma, St. Louis, MO, USA), cholera toxin (Sigma, St. Louis, MO, USA), and hydrocortisone (Sigma, St. Louis, MO, USA) [23 (link)]. Stable T8 cell lines with hDsg3.myc (D3) and the matched empty vector control line (Vect) were generated in this laboratory following the procedures described previously [24 (link),25 (link)]. For the dose and time-course experiments with verteporfin (VP), cells were seeded at approximately 70~80% confluent densities and cultured in KGM [23 (link)] in the presence and absence of VP at various concentrations (with a range of dosages similar or less than others [8 (link),9 (link),26 (link)]) for different time frames before analyses by different techniques and assays, respectively.

Blood samples were collected by venepuncture into vacutainers containing heparin (Sastedt AG, Germany). Complete blood differential counts were performed in whole blood using a haematology analyser (Sysmex XN-1000, Sysmex, Germany). Whole blood count data for mild COVID-19 infection patients were unavailable as they were not hospitalised. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Paque™ PLUS (GE Healthcare, UK) of diluted blood (1:1) in RPMI 1640 medium (Sigma Aldrich, Poole, UK), and overlayered blood was centrifuged for 30 min at 400 × g at 20 °C without brake [71 (link)]. Isolated PBMCs were frozen by resuspending cells in a freezing medium consisting of 10% DMSO (Sigma Aldrich) in heat-inactivated fetal calf serum (FCS; Biosera, UK) and stored at -80°C until further analysis.

0.5 million freshly isolated BAL cells were cultured in DMEM, high glucose, with stable glutamine and sodium pyruvate (Biosera) supplemented with 2% Fetal Calf Serum (FCS) (Biosera) and 1x concentration of penicillin-streptomycin (from 100x concentrated solution, Biosera) in a humidified incubator at 37°C containing 5% CO₂ for 30min, with washes to remove non-adherent cells, such as lymphocytes and eosinophils. In order to avoid changes in gene expression caused by the contact of cells with rigid surfaces that may lead to detectable altered protein levels of COL1A1 or OPN, cells were cultured for a short period of thirty minutes. Cells were fixed with 4% formaldehyde (FA) for 20 min at RT and were stored overnight in 1% FA.