Trypsin ethylenediaminetetraacetic acid edta solution

Human fibrinogen and BAY 11-7082 ((E)3-[(4-Methylphenyl)sulfonyl]-2- propenenitrile) were purchased from Calbiochem (La Jolla, CA, USA); Ham's F12K medium was from Gibco-Invitrogen (Carlsbad, CA, USA). Collagen type I, fetal bovine serum and trypsin/ethylenediaminetetraacetic acid (EDTA) solution were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Recombinant TNF-α and ELISAs for interleukin-8 (IL-8), interleukin-1β (IL-1β) and Platelet Factor 4 (PF4) determination were from R&D Systems (Minneapolis, MN, USA). ACD Vacutainers® were used for blood collection (BD Brasil, Sao Paulo, Brazil). All other products were from Sigma-Aldrich unless otherwise stated.

The human breast adenocarcinoma cell lines MCF-7 and MDA-MB-231 were obtained from the European Collection of Cell Cultures (ECCC, Salisbury, UK). These cell lines were grown in 100-mm Petri dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin in an air-CO₂ (95:5) atmosphere at 37°C. Confluent cells were washed twice with phosphate-buffered saline (PBS) and harvested by a brief incubation with trypsin- ethylenediaminetetraacetic acid (EDTA) solution (Sigma-Aldrich, St. Louis, MO, USA). Geneticin (G418), culture medium, and sera were purchased from Invitrogen Life Technologies, Carlsbad, CA, USA. Immobilon-P membranes were from Millipore (Merck Millipore, Billerica, MA, USA). Mytomycin C, MTT, puromycin, and hygromycin B were from Sigma-Aldrich.

Human neuroblastoma SH-SY5Y cells were purchased from ATCC (Manassas, VA, USA) and maintained at 37 °C in a humidified atmosphere supplied with 5% CO₂ in a Dulbecco’s Modified Eagle Medium (DMEM):Ham’s F12 medium (1:1) (Gibco^®, Life Technologies, Carlsbad, Canada) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, and 100 U/mL penicillin and 100 g/mL streptomycin (Lonza, Basel, Switzerland). Cell harvesting was performed using a Trypsin/Ethylenediaminetetraacetic acid (EDTA) solution (Sigma-Aldrich, Steinheim, Germany).
The cells were seeded in 60 mm dishes (1.5 × 10⁶ cells/dish) and transfected with pHM6-αSyn-wt (#40824, AddGene, Cambridge, MA) or pHM6-Mock using a K2^® Transfection System (Biontex Laboratories GmbH, München, Germany) to generate SH-SY5Y stably overexpressing αSyn (SH-Syn) or control cells (SH-Mock), respectively. G418 at 800 μg/mL (Sigma-Aldrich, Steinheim, Germany) was added to the culture medium 48 h after transfection for transfected cell selection; then, the cells were maintained in a culture medium supplemented with G418 at 200 μg/mL.
Cell morphology phase-contrast images were acquired via an inverted microscope Zeiss Axiovert 200 (Carl Zeiss, Gottingen, Germany) equipped with a color digital camera Axiocam MR (Carl Zeiss, Gottingen, Germany) using the Axiovision 4.8 program (Vysis, Downers Grove, IL, USA).

ASP (Sigma, St. Louis, MO, USA); MET (European Pharmacopeia, Strasbourg, France); ³H-Deoxy-D-glucose (2-[1,2-³H(N)]-deoxy-D-glucose: specific activity 60 Ci/mmol); ¹⁴C-n-butyric acid ([1-¹⁴C-n-butyric acid]; specific activity 55 mCi/mmol) (American Radiolabeled Chemicals Inc., St Louis, MO, USA); ³H-thymidine ([methyl-³H]-thymidine; specific activity 79 Ci/mmol) (GE Healthcare GmbH, Freiburg, Germany); trypsin-EDTA 0.25% (PAN-Biotech™, Aidenbach, Germany); crystal violet, triton X-100, trichloroacetic acid (TCA) (Merck, Darmstadt, Germany); Dulbecco’s Modified Eagle Medium (DMEM; catalogue #D5796), Minimum Essential Medium (MEM; catalogue #M-0643), antibiotic/antimycotic solution (100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B), bovine serum albumin (BSA), fetal bovine serum (FBS), reduced nicotinamide adenine dinucleotide (NADH), resazurin, hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES), sodium pyruvate; sulforhodamine B (SRB), tris-HCl (tris(hydroxymethyl)-aminomethane hydrochloride), trypsin–ethylenediaminetetraacetic acid (EDTA) solution (Sigma, St. Louis, MO, USA).

TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program (Cat. no. 8129). This cell line is derived from a clone of HeLa cells designed for expressing HIV-1 virus entry receptor and co-receptors (CD4, CXCR4, and CCR5) with an integrated luciferase gene under the control of the HIV-1 promoter long terminal repeat (LTR) [16 (link),17 (link)], which makes it possible to measure infection. HEK293T cells were purchased from the American Tissue Culture Collection (ATCC). TZM-bl and HEK293T cells were cultured at 37 °C with 5% CO₂ in DMEM supplemented with heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA). Cell monolayers were detached via treatment with a Trypsin ethylenediaminetetraacetic acid (EDTA) solution of 0.25% (Sigma-Aldrich, St. Louis, MO, USA).
H9/HTLV-IIIB cells (ATCC-CRL-8543) were persistently infected and were used to produce HIV-1IIIB (X4-tropic HIV-1) viral stocks. Human peripheral blood mononuclear cells (PBMC) from two healthy donors were obtained from the blood bank of the Hospital Universitario San Vicente Fundación. PBMC were isolated through a density gradient with Ficoll-Histopaque (Sigma-Aldrich, St. Louis, MO, USA). H9/HTLV-IIIB and PBMC were maintained in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% heat-inactivated FBS incubated in a humid atmosphere with 5% CO₂ at 37 °C.

NOK-si (kindly provided by Professor Carlos Rossa Jr., from the Cellular and Molecular Biology Laboratory, Department of Periodontics, School of Dentistry, São Paulo State University—UNESP) and FGH (Rio de Janeiro Cell Bank; code: 0089) cells were thawed and cultured in Eagle medium modified by Dulbecco high glucose (DMEM) (4.5 g/L), supplemented with 2.0 mmol·L⁻¹ of glutamine (Lonza, Basel, Switzerlnad, 10% of bovine serum), 1% antibiotic/antimycotic (penicillin G—10,000 μg·mL⁻¹, streptomycin—10,000 μg·mL⁻¹, amphotericin B—25 μg·mL⁻¹) (Sigma-Aldrich, St. Louis, MO, USA) incubated with 5% CO₂ in the atmosphere and cultured until reaching 80% confluence. The cells were washed (PBS buffer; pH 7.2), detached from the plate with a 0.05% trypsin ethylenediaminetetraacetic acid (EDTA) solution (Sigma-Aldrich, St. Louis, MO, USA), and then the cells were counted. In the experiments, 2.5 × 10⁵ cells/well were used for NOK-si and 1.2 × 10⁵ cells/well were used for FGH. For all experiments, cells between the 3rd and 8th passages were used [28 (link)].