Dmem containing 4.5 g l glucose

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DMEM (Dulbecco's Modified Eagle Medium) containing 4.5 g/L glucose is a basal medium used for the in vitro culture of cells. It provides the necessary nutrients, including amino acids, vitamins, and other essential components, to support cell growth and maintenance.

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4.5 g/L glucose DMEM-glucose L-glucose L-DMEM

6 protocols using dmem containing 4.5 g l glucose

Isolation and Culture of Human Liver Cells

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This study was approved by the ethics committee of our institution. Liver tissue was obtained from a Ministry of Health-approved hospital tissue bank (Table 1). ADHLSCs were obtained after primary culture of the parenchymal fraction cells, as previously described [14 (link), 17 (link)]. The supernatant, containing the non-parenchymal cell fraction, was processed by Nycodenz gradient (Myegaard, Oslo, Norway) centrifugation to isolate HSCs [26 (link)]. Both cell types were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS; Life Technologies) and 1% penicillin/streptomycin (Life Technologies), at 37 °C in a humidified atmosphere containing 5% CO₂, as previously described [17 (link)]. When the cells reached 80% confluence, they were detached by treatment with 0.05% Trypsin-EDTA (Life Technologies). The viability of the recovered cells was evaluated using the trypan blue exclusion assay.Table 1

Characteristics of the four liver donors whose samples were used for the isolation of HSCs and ADHLSCs used in the current study

Donor number	Age	Gender	Cause of death	Blood group	Ischemia time
89	3 days	M	Respiratory	A+	4 h
93	2 years	F	Metabolic disease (liver transplanted)	O+	1 h 43 min
98	7 days	M	Cardio-respiratory arrest	O-	4 h 20 min
105	46 years	F	Trauma	B+	9 h 43 min

Najimi M., Berardis S., El-Kehdy H., Rosseels V., Evraerts J., Lombard C., El Taghdouini A., Henriet P., van Grunsven L, & Sokal E.M. (2017). Human liver mesenchymal stem/progenitor cells inhibit hepatic stellate cell activation: in vitro and in vivo evaluation. Stem Cell Research & Therapy, 8, 131.

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Isolation and Culture of Human Hepatic Stem Cells

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The present study was accepted by the institution ethical review board for the use of human-derived tissue, under appropriate informed consent of tissue donors. An agreement from the Belgian Ministry of Health was delivered for hepatocytes and hepatic stem cell isolation and banking.
Liver cell suspensions were recovered after a two-step collagenase perfusion technique of livers from healthy cadaveric donors. Following filtration and low-speed centrifugation, the parenchymal fraction, predominantly constituted by hepatocytes, was recovered and seeded as primary cultures. ADHLSCs were then obtained as previously described (Najimi et al. [2 (link)]). The cells were cultured using DMEM containing 4.5 g/l glucose (Life Technologies) supplemented with 10% fetal calf serum (FCS) (Life Technologies) and 1% penicillin/streptomycin (Life Technologies), at 37°C in a fully humidified atmosphere (5% CO2). When reaching 80% confluence, cells were lifted with 0.05% Trypsin-EDTA (Life Technologies) and seeded at a density of 5000 cells/cm². The viability of recovered cells was evaluated using Trypan blue exclusion assay at each passage.

El-Kehdy H., Sargiacomo C., Fayyad-Kazan M., Fayyad-Kazan H., Lombard C., Lagneaux L., Sokal E., Najar M, & Najimi M. (2017). Immunoprofiling of Adult-Derived Human Liver Stem/Progenitor Cells: Impact of Hepatogenic Differentiation and Inflammation. Stem Cells International, 2017, 2679518.

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Evaluating Collagen Secretion in HSCs

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After removal of the inserts containing ADHLSCs and aspiration of the medium, recovered HSCs were washed with sterile PBS and incubated in serum-free medium (DMEM containing 4.5 g/L glucose [Life Technologies]) for 24 h. Then, the supernatant was collected, and cells were detached for counting and evaluation of viability. Collagen secretion was evaluated using an ELISA kit for procollagen type I C-Peptide (Takara Bio Inc., Shiga, Japan). The absorbance at 450 nm was measured with a VICTOR X4 Plate Reader (Perkin Elmer, Waltham, MA, USA). The results were randomized according to the number of cells collected.

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Emulsion Transfer and In Vitro Digestion

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(all-E)-lutein (96%) used for emulsion transfer experiments and in vitro digestions was purchased from CaroteNature (Mussingen, Switzerland). (all-E)-lutein (70% pure) for piglet experiments was from Naturex (Avignon, France). Sunflower oil (Lesieur, France) and canned spinach (Casino, France) were food grade. Sodium acetate and formic acid (HCOOH) were purchased from Merck (Lyon, France). Apo-8'-carotenal (96% pure, used as an internal standard), triolein (65% pure), L-α-phosphatidylcholine and bovine serum albumin (BSA), porcine bile extract and pancreatin were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). DMEM containing 4.5 g/L glucose, trypsin-EDTA (500 mg/L and 200 mg/L, respectively), non-essential amino acids, penicillin/streptomycin and PBS were from Life Technologies (Villebon sur Yvette, France). Foetal bovine serum (FBS) was from Dutscher (Brumath, France). Deionized H 2 O was purified by a Milli-Q Water Purification system (Millipore, MA, USA). All solvents used were HPLC grade from either SDS (Peypin, France) or Merck.

Margier M., Buffière C., Goupy P., Remond D., Halimi C., Caris-Veyrat C., Borel P, & Reboul E. (2018). Opposite Effects of the Spinach Food Matrix on Lutein Bioaccessibility and Intestinal Uptake Lead to Unchanged Bioavailability Compared to Pure Lutein. Molecular nutrition & food research, 62(11).

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DENV2 WT and NS1:T164S Mutant Virus Production

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Vero cells (green African monkey kidney epithelial cells, ATCC) were cultured in DMEM containing 4.5 g/L glucose (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (P/S) at 37°C in 5% CO₂. Expi293F cells were cultured in serum-free Expi293 Expression Medium at 37°C in 8% CO₂. The infectious clone-derived DENV2 WT (GenBank accession: EU081177) and NS1:T164S mutant viruses used in this study were described previously (Chan et al., 2019 (link)). The anti-NS1 56.2 IgG antibody (Ab56.2) was obtained from the hybridoma cell culture as described previously (Rozen-Gagnon et al., 2012 (link)). Briefly, the Ab56.2 hybridoma cells were grown in PFHM II medium (Gibco) at 37°C in 5% CO₂. Culture supernatants were collected every 4 d, clarified, and filtered through 0.2 μm filter membrane. Ab56.2 was purified from the supernatant through a Protein G HiTrap column (GE Healthcare) using the AKTA purification system (Cytiva). The bound Ab was eluted from the Protein G column using 0.1 M glycine (pH 2.7), neutralized with 1 M Tris-HCl (pH 9.0), and dialyzed with PBS for storage at –30°C until use. Cell lines were tested negative for mycoplasma contamination.

Chew B.L., Ngoh A.Q., Phoo W.W., Chan K.W., Ser Z., Tulsian N.K., Lim S.S., Weng M.J., Watanabe S., Choy M.M., Low J., Ooi E.E., Ruedl C., Sobota R.M., Vasudevan S.G, & Luo D. (2024). Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein. eLife, 12, RP90762.

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Wnt/Cdc42 Signaling in Rat ADSC Differentiation

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Anti-Cdc42 antibody was purchased from Abcam (Cambridge, MA, USA) and anti-Dvl-2 antibody was purchased from Cell Signaling Technology (Trask Lane Danvers, MA, USA). Anti-Insulin, anti-Ngn3, anti-NeuroD1, anti-PDX1, anti-non-p-GSK3β, anti-p-GSK3β, anti-non-p-β-catenin, and anti-β-actin antibodies were purchased from Affinity Biosciences (Cincinnati, OH, USA). HPR-labeled anti-rabbit IgG of goat and HPR-labeled anti-rat IgG of goat were purchased from Zhongshan Jinqiao Company (Beijing, China). Recombinant human Wnt-3a was purchased from R&D Inc. (Minneapolis, MN, USA), and Cdc42 inhibitor ML141 (5mg) was purchased from Selleck (Houston, TX, USA). DTZ was obtained from Sigma (St. Louis, MO, USA). Bovine serum albumin (BSA), Triton-100 and DMSO were purchased from Solarbio (Beijing, China). Low-glucose Dulbecco’s modified Eagle’s medium (DMEM) and DMEM containing 4.5 g/L glucose were obtained from Gibco-BRL (Gaithersburg, MD, USA). Wistar rat ADSC basal medium was purchased from Cyagen (USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Cromwell, CT, USA). Penicillin and streptomycin were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Krebs-Ringer bicarbonate HEPES (KRBH) was purchased from PanEra Company (Guangzhou, China). Enzyme-linked immunosorbent assay (ELISA) Kit for Insulin was purchased from Cloud-Clone Corp (Wuhan, China).

Xiao X.H., Huang Q.Y., Qian X.L., Duan J., Jiao X.Q., Wu L.Y., Huang Q.Y., Li J., Lai X.N., Shi Y.B, & Xiong L.X. (2019). Cdc42 Promotes ADSC-Derived IPC Induction, Proliferation, And Insulin Secretion Via Wnt/β-Catenin Signaling. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 12, 2325-2339.

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