Trypsin ethylenediaminetetraacetic acid

Monocyte-derived DC from healthy donors were cultured and induced to maturation as described [24 (link),32 (link)]. Purity of DC was always ≥99%. Tat-mediated Env entry in monocyte-derived DC was assessed as described [32 (link)]. Briefly, sera were diluted 1:30 in phosphate buffered saline and incubated for 60 min at 37°C with trimeric Env (0.4 μM) previously premixed for 10 min at 25°C with Tat (0.4 μM) or degassed PBS (control). Samples were then added to DC (2×10⁵ cells/mL) to a 1:5 final dilution and incubated for 10 min at 37°C. Cells were then washed with cold medium and treated for 10 min at 37°C with trypsin-ethylenediaminetetraacetic acid (Life Technologies, Paisley, UK) to remove any externally bound protein. After fixation and permeabilization, DC were stained with rabbit anti-gp120 polyclonal Abs (Chem Progress, Milan, Italy) or purified rabbit IgG control Abs (Sigma-Aldrich, Milan, Italy), followed by FITC-conjugated anti-rabbit Ig (Pierce, Rockford, IL, US). Fluorescence was analyzed by flow cytometry, and results expressed as the percentage of Env positive cells as compared to isotype stained samples. A pool of sera from six healthy donors and a monoclonal Ab against Tat (mAb 2A4.1, obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) were used as negative and positive controls, respectively.

The skin tissues were treated with RNAlater, (Life Technologies, Carlsbad, CA, USA) followed by liquid nitrogen refrigeration were collected. The cultured cells were digested by 0.25 % trypsin-ethylene diaminetetraacetic acid (Life Technologies) and washed twice with PBS before homogenizing. Total RNA was extracted using TRIzol (Life Technologies) according to the manufacturer’s instructions. Relative BDKRB1/Bdkrb1 mRNA expression levels were analyzed by qRT-PCR with double-stranded, DNA-binding dyes as reporters, using GoTaq qPCR Master Mix (Promega, Madison, WI, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was used as the internal reference. RT-PCR primers were: Bdkrb1 forward: 5’-CCCAACTACAGTTGTGAACGC-3’, Bdkrb1reverse: 5’-AGGATGATGCCATGCACAGT-3’; Gapdh forward: 5’-CTCCTCCTGTTCGACAGTCAGC-3’; Gapdh reverse: 5’-CCCAATACGACCAAATCCGTT-3’; BDKRB1 forward: 5’-GGCAGCTTCTGATCTGGTGT-3’, BDKRB1 reverse: 5’-GTAGCGGTCCTGACTGATGG-3’; GAPDH forward: 5’-GTGTTCCTACCCCCAATGTGT-3’; GAPDH reverse: 5’-TGAAGTCGCAGGAGACAACC-3’.

Hep3B and HepJ5 cells were seeded as 5 × 10⁵ cells per 6 cm dish, incubated overnight, and then treated with 1 or 2 mg/mL TCEE, respectively, for 24 hr. After treatment, cells were washed by phosphate buffered saline (PBS) and harvested by 0.05% trypsin-ethylenediaminetetraacetic acid (Life Technologies, Carlsbad, CA, USA). Cell pellets were collected by centrifuge and resuspended in 1 mL PBS with 4 mL 75% ethanol in −20°C overnight for cell fixation. Fixed cells were centrifuged and washed by 5 mL PBS in room temperature. Before analysis, cell suspensions were mixed with 1 mL propidium iodide buffer alone (RNase A 0.2 mg/mL, triton X-100 0.1%, and 20 μg/mL propidium iodide, PI) or in combination with the FITC-labelled annexin V binding buffer (Invitrogen, Life Technologies, Carlsbad, CA, USA) in room temperature for 15 min staining. Stained cells were finally measured by the FACScan flow cytometer (BD Biosciences, CA, USA) and analyzed by the CellQuest software (BD Biosciences, CA, USA).

1,2-Hydrogenated-L-α-phosphatidylcholine (HSPC) was from Northern Lipids Inc, Vancouver, Canadá. CUR, cholesterol (Chol), Tween 80, (6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), 3-(4,5-dimetiltiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), phorbol 12-13-acetate (PMA), Mowiol, 2-mercaptoethanol, lipopolysaccharide (LPS) and dexamethasone 21-phosphate disodium salt (DEX) were from Sigma-Aldrich (MO, USA). Hoechst 33342, CellMask Deep Red Plasma membrane stain, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (carboxy-H2DCFDA), Lucifer Yellow (LY), Roswell Park Memorial Institute 1640 (RPMI), Modified Eagle Medium (MEM), penicillin-streptomycin sulphate, glutamine, sodium pyruvate and trypsin/ethylenediamine tetra acetic acid were from Life Technologies (NY, USA), fetal bovine serum (FBS) was from Internegocios (Buenos Aires, Argentina). The other reagents were of analytic grade from Anedra, Research AG (Buenos Aires, Argentina).

MCF-7 (RIKEN BRC, Saitama, Japan) and NHDF cells (Cosmo Bio Co., Ltd., Tokyo, Japan) were cultured in Dulbecco’s modified Eagle’s medium (11965092; Thermo Fisher Scientific Inc.) supplemented with 10% fetal bovine serum (Funakoshi Co., Ltd., Tokyo, Japan) and 1% penicillin (15140122; Thermo Fisher Scientific Inc.) at 37 °C under 5% CO₂. Cells were detached using 0.05% trypsin-ethylenediaminetetraacetic acid (25300; Life Technologies, Carlsbad, CA, USA). Except for the experiments, the cells were cultured in conventional plastic culture dishes.
To demonstrate the cell adaptability (Fig. 2E to H), 1.0 × 10⁶ cells were seeded into the metallic culture vessels and cultured for 24 h, while cells with the same density on culture surfaces were seeded and cultured in 35-mm plastic culture dishes. After culturing, the supernatant was collected to count the number of cells detached from the culture surface. To measure cell viability, live and dead cells were stained with calcein-AM and propidium iodide, respectively. The ratios of live-cell areas were used to determine cell viability.