Penicillin streptomycin

HEK293T and Jurkat cell lines (ATCC) were maintained either with DMEM or RPMI1640 medium containing 10% of FBS, penicillin/streptomycin antibiotics, and l-glutamine.
For the measurement of target lysis, K562 cells were first cultured overnight in RPMI1640 without phenol red (Gibco) enriched with 10% FBS, non-essential amino acids (NEAA, Gibco), l-glutamine (Life Technologies) and Sodium Pyruvate (Eurobio). Prior killing assay, K562 cells were diluted at 1 × 10⁶/mL in a culture medium supplemented with 2.5 mM Probenecid (Invitrogen) and stained with calcein-AM (Invitrogen) for 30 min. Then, the excess calcein was washed out.
For in vitro cell treatment, PBMCs were resuspended in RPMI 1640 supplemented with 10% human serum (EFS, Etablissement Français du Sang), 1% Penicillin–Streptomycin (Dutscher), l-glutamine (Life Technologies), Sodium Pyruvate (EuroBio) and HEPES (EuroBio).

In vitro cellular cholesterol efflux induced by HDL was determined using [3H]cholesterol-labeled J774A.1 mouse macrophages (ATCC^® TIB67™, Manassas, VA, USA), as previously described [31 (link)]. Briefly, 1.5 × 10⁵ cells/well were seeded in 6-well plates and allowed to grow for three days in RPMI 1640 medium containing 2 mM L-Glutamine (Pan Biotech, Aidenbach, Germany) complemented with 10% fetal bovine serum (FBS) (Pan Biotech) and 100 U/mL penicillin/streptomycin (Dominique Dutscher, Brumath, France). After that, cells were labeled for 60 h in the presence of one µCi/well of [1α,2α(n)-3H]cholesterol (GE Healthcare, Little Chalfont, UK) and 5% FBS. The cells were then equilibrated with 0.2% bovine serum albumin (BSA) in medium overnight and incubated for 4 h with mice HDL (25 µg/mL protein), previously isolated by ultracentrifugation and dialyzed in phosphate-buffered saline, as described above. Radioactivity was measured in both the medium and the cells, and the percentage of cholesterol efflux was calculated.

As validated and described [27 (link),30 (link)], HUVECs were purchased from PromoCell and cultivated according to the manufacturer’s recommendations. Endothelial cell growth medium (PromoCell) containing 2% (v/v) fetal calf serum (FCS), 0.4% (v/v) endothelial growth supplement: 0.1 ng/mL human EGF, 1.0 μg/mL hydrocortisone, 1 ng/mL human bFGF, 90 µg/mL heparin, and 1% (v/v) penicillin/streptomycin (Dutscher, Brumath, France) were used in a completely humid atmosphere at 37 °C and 5% CO₂. The confluent cells were detached using the PromoCell detachment kit containing 30 mM Hepes, trypsin/EDTA solution (0.04%/0.03%), and trypsin neutralizing solution. The FCS was reduced to 1% 24 h before the experiment. All experiments were performed on subconfluent monolayer endothelial cells (80%) after the third passage.

HeLa cytosols (88882) and Rabbit Reticulocyte lysates (L4540) were respectively purchased from Thermofisher and Promega. Human Embryonic Kidney (HEK) and rat insulinoma INS-1E cell lines were respectively obtained from ATCC and MTA, Professor Maescler’s laboratory (RRID:CVCL_0351). Both cell lines were routinely maintained in our laboratory. HEK cells were maintained in DMEM (Gibco) media with 25 mM glucose, 1 mM pyruvate, 2 mM Glutamax (Gibco), 10% SVF, and 1% penicillin/streptomycin (Dutscher). INS-1E cells were maintained in RPMI 1640 (Gibco) with 1 mM pyruvate, 2 mM Glutamax, 10 mM HEPES buffer, 50 µM β-mercaptoethanol, 5% SVF, and 1% penicillin/streptomycin. % refer to volume proportions.

HT-29 human epithelial cells were used for immunomodulation assays; either the parental lineage (HT-29, colon adenocarcinoma; ATCC HTB-38) or a lineage transfected with the secreted alkaline phosphatase (SEAP) reporter gene for NF-kB activation monitoring (HT-29/kb-seap-25) (Lakhdari et al., 2010 (link)). The reporter HT-29/kb-seap-25 cells were cultured in RPMI-Glutamine medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Corning), 1% non-essential amino acids, 1% sodium pyruvate, 1% HEPES buffer (Thermofisher Scientific), and 1% penicillin-streptomycin (Lonza) according to Lakhdari et al. (2010) (link). The parental HT-29 cells were cultured in high-glucose DMEM medium (Dominique Dutscher) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Do Carmo et al., 2017 (link)). For subcultures, cells were rinsed with DPBS (Thermofisher Scientific) and detached with a trypsin (0.05%) – EDTA (0.02%) solution (Sigma). Periodically, 100 μg/mL Zeocin (Invivogen) was applied to the HT-29/kb-seap-25 cell culture in order to maintain selective pressure on the cells containing the transfected plasmid.

Human embryonic kidney cell line HEK293T (ATCC) and the kidney epithelial cell line Vero (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Dutscher, Issy-les-Moulineaux, France) or modified Eagle’s medium (MEM, Dutscher) supplemented with 10% fetal bovine serum heat inactivated (FBS, Dutscher) and completed with 2 mmol.L^-1l-glutamine (Dutscher), 100 U.mL^-1–0.1 mg.mL^-1 penicillin-streptomycin (Dutscher), 1 mmol.L^-1 sodium pyruvate (Dutscher) and 250 μg.mL^-1 amphotericin (Dutscher). Cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C in Petri dishes.

The MC3T3-E1 cells were plated in expansion medium composed of alpha-Modified Eagle Medium (α-MEM) (Dutscher) containing 10% FCS, 1% penicillin/streptomycin and 1% glutamine (Dutscher). Osteogenesis differentiation was started when MC3T3 had reached confluence (day 0) by incubation with osteogenic inductors (50 μg/ml ascorbic acid and 10 mM β-glycerophosphate (Sigma-Aldrich Corporation) for 14 days.