Human primary HSC (hHCS) were isolated from fragments of normal livers obtained from optimal cadaveric liver donors, as previously described [31 (link),32 (link)]. Human immortalized HSCs (LX2) were a kind gift from Dr. Friedman (Mount Sinai School of Medicine, New York, NY, USA). LX2 cells and hHSCs were routinely grown in DMEM culture medium (Lonza, Basel, Switzerland) supplemented with 10% foetal bovine serum (FBS) (Lonza), and 100 U/ml penicillin and streptomycin (Sigma-Aldrich). Human monocyte-derived macrophages (HMDMs) were differentiated from peripheral blood monocytes by incubation in RPMI and 10% FBS (Lonza) for 7 days, as previously described [33 (link)]. HSCs were serum-starved overnight before the following treatments were performed: 10 ng/ml TGFB1 (Preprotech), 1 μg/ml rCD5L, 25 ng/ml PDGF (Preprotech) and/or 1 μg/ml human Albumin (hSA) (Grífols, Barcelona, Spain). In these experiments, the concentration of rCD5L was chosen based on previous dose-response studies on macrophages [27 (link)]. All cells were grown at 37 °C and 5% CO2.
Dmem culture medium
Manufactured by Lonza
Sourced in Switzerland, Belgium
DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium commonly used in laboratories for the growth and maintenance of a variety of cell lines. It provides essential nutrients, amino acids, vitamins, and other components required for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Protein ladders, by Santa Cruz Biotechnology (2 mentions)
Power sybr green, by Thermo Fisher Scientific (2 mentions)
Bca assay kit, by Merck Group (2 mentions)
Pre designed sirna, by Thermo Fisher Scientific (2 mentions)
Polyplus interferin, by Source Bioscience (2 mentions)
Protein a g agarose gel beads, by Santa Cruz Biotechnology (2 mentions)
Rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Lonza (2 mentions)
Streptomycin, by Lonza (2 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Tgfb1, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Lonza (1 mentions)
Sp5 aobs, by Leica (1 mentions)
Hoechst 34580, by Thermo Fisher Scientific (1 mentions)
Attofluor cell chamber, by Thermo Fisher Scientific (1 mentions)
Be17 605e, by Lonza (1 mentions)
De14 801f, by Lonza (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
9 protocols using dmem culture medium
1
Isolation and Culture of Human Hepatic Stellate Cells
2
Imaging Neutrophil Extracellular Trap Formation
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Neutrophils were stained for DNA with Hoechst 34580 (diluted 1:10,000, Life Technologies) and for extracellular DNA with PI (diluted 1:400, Sigma Aldrich) in 500 µl DMEM culture medium (Biowhittaker, Lonza). The cells were allowed to attach to gelatin-coated coverslips at 37°C for at least 1 h.
To induce NETosis, 500 µl 2 × 1010 bacteria/ml were added to 500 µl 2 × 107 neutrophils/ml in a Attofluor Cell Chamber (Thermo Fisher Scientific, Bleiswijk, The Netherlands). The chamber was sealed, and the neutrophils were continuously imaged with a confocal microscope (Leica SP5 AOBS) with a 40× magnification and numerical aperture (n.a.) of 1.25. Hoechst and PI were excited by 405 nm (emission BP 450–550 nm) and 561 nm (emission 570–620 nm) lasers, respectively. NETs were visible as PI positive elongated structures and were quantified (seeNETs Quantification ).
In order to study the effect of Protein A on NETosis induction by S. aureus Newman ΔSpA ΔSbi strain, 100 µl of either 0.01, 0.1, or 1 mg/ml of purified Protein A (Sigma Aldrich, Zwijndrecht, The Netherlands) was added to the Newman ΔSpA ΔSbi strain prior to co-incubation with neutrophils (final concentration range 0.9–90 µg/ml Protein A).
To induce NETosis, 500 µl 2 × 1010 bacteria/ml were added to 500 µl 2 × 107 neutrophils/ml in a Attofluor Cell Chamber (Thermo Fisher Scientific, Bleiswijk, The Netherlands). The chamber was sealed, and the neutrophils were continuously imaged with a confocal microscope (Leica SP5 AOBS) with a 40× magnification and numerical aperture (n.a.) of 1.25. Hoechst and PI were excited by 405 nm (emission BP 450–550 nm) and 561 nm (emission 570–620 nm) lasers, respectively. NETs were visible as PI positive elongated structures and were quantified (see
In order to study the effect of Protein A on NETosis induction by S. aureus Newman ΔSpA ΔSbi strain, 100 µl of either 0.01, 0.1, or 1 mg/ml of purified Protein A (Sigma Aldrich, Zwijndrecht, The Netherlands) was added to the Newman ΔSpA ΔSbi strain prior to co-incubation with neutrophils (final concentration range 0.9–90 µg/ml Protein A).
3
Culturing Human Cancer Cell Lines
4
Biochemical Assays and Cell Culture Techniques
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Chemical reagents for preparing buffers and the BCA Assay Kit were purchased from Sigma Aldrich, U.K. and Fisher Scientific, U.K. Buffers used include RIPA buffer, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), blocking buffer, cell lysis buffer, elution buffer, SDS sample buffer, and ECL detection reagent [23 ]. PolyPlus INTERFERin was purchased from Source Bioscience, U.K. Pre-designed siRNA, Opti-MEM, Power SYBR Green, RNA to cDNA kit, and gel casting materials were purchased from Life Technologies, U.K. Antibodies, Protein A/G agarose gel beads, and protein ladders were obtained from Santa Cruz Biotechnology, UK and Cell Signalling Technologies, U.K. Recombinant mouse proteins, ELISA antibodies and detection reagents were purchased from Peprotech Ltd. DMEM culture medium and other cell culture materials were purchased from Lonza, U.K. PCR primers were designed with Primer3 Plus Bioinformatics Software and NCBI BLAST and purchased from Eurofins Genomics.
5
Culturing Human Melanoma and Fibroblast Cells
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Human melanoma cell line A375 (provided by the Laboratory of Cell Pathology, Faculty of Biotechnology, University of Wrocław, Poland) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) culture medium (Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland) supplemented with 4 mM glutamine (Life Technologies, Carlsbad, CA, USA) and 5% fetal bovine serum (Life Technologies). Human melanoma cell line Hs294T (provided by the Institute of Immunology and Experimental Therapy) was cultured in DMEM culture medium (Lonza, Basel, Switzerland) supplemented with 2 mM glutamine (Life Technologies) and 5% fetal bovine serum (Life Technologies). The normal human dermal fibroblast (NHDF; Lonza) cell line was cultured in MEMα culture medium (Lonza) supplemented with 2 mM glutamine (Life Technologies) and 10% fetal bovine serum (Life Technologies). All the media contained 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 μg/mL amphotericin B (Lonza). The cells were cultured at 37°C in a humid atmosphere saturated with 5% CO2.
6
Caco-2 Cell Culture Protocol
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Adherent human epithelial colorectal adenocarcinoma cells (Caco-2; ATCC® HTB-37™), were used at passage numbers 25–40. They were cultured and maintained in 75 cm2 cell culture flasks (Corning®; New York, USA) at 37 °C in a humidified 5% CO2 atmosphere (HERAcell 240 incubator; Marietta, USA). Complete cell culture medium was prepared by supplementing Dulbecco’s Modified Eagle Medium (DMEM) culture medium (LONZA; Verviers, Belgium) with 10% (v/v) heat inactivated Foetal Bovine Serum (FBS) (Gibco®, Life technologies; New York, USA), 1% (v/v) of Penicillin–Streptomycin (Sigma-Aldrich; Steinheim, Germany), and 1% (v/v) of MEM Non-Essential Amino Acids (NEAA) (Gibco®, life technologies; New York, USA). The complete medium is further referred to as DMEM+.
7
Flow cytometry optimization via orbital shaking
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In order to submit a larger number of cells to flow, orbital shaker experiments were performed using the MS1 (MILE SVEN1) cell line (ATCC®CRL2279TM, Les Ullis, France). Cells were maintained in a DMEM culture medium (Lonza, Basle, Switzerland) and supplemented with 5% v/v fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, in a humid atmosphere of 37 °C with 5% CO2. Cells were then placed at 37 °C as control and cells under flow were placed under agitation of our laboratory digital orbital shaker for 24 or 72 h at 210 rpm (12 dyn/cm2) or 260 rpm [36 (link)].
In order to verify that cells were submitted to laminar (protective) flow we measured Nos3 (encoding for eNOS) and Edn1 (encoding for ET-1) gene expression. Laminar flow has been shown to increase eNOS epression and to decrease Edn1 expression whereas a disturbed flow has the opposite effect [37 (link),38 (link),39 (link),40 (link),41 (link)].
Cells were then collected for transcriptomic analyses as described below.
In order to verify that cells were submitted to laminar (protective) flow we measured Nos3 (encoding for eNOS) and Edn1 (encoding for ET-1) gene expression. Laminar flow has been shown to increase eNOS epression and to decrease Edn1 expression whereas a disturbed flow has the opposite effect [37 (link),38 (link),39 (link),40 (link),41 (link)].
Cells were then collected for transcriptomic analyses as described below.
8
Cultivation of Endothelial Cell Lines
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Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from Lonza (Basle, Switzerland) and cultivated in endothelial cell growth medium-2 (EGM-2, Lonza, Basle, Switzerland) supplemented with the EGMTM-2 SingleQuots kit (Lonza, Basle, Switzerland), at 37 °C and 5% CO2. Cells from passages 1 to 5 were used for experiments. For orbital shaker experiments, the MS1 (MILE SVEN1) cell line was used (endothelial C57BL6 cell line obtained from the islets of Langerhans, ATCC®CRL2279TM, Les Ulis, France). Cells were maintained in a DMEM culture medium (Lonza, Basle, Switzerland) supplemented with 5% fetal bovine serum (Eurobio scientific, Les Ulis, France), glutamine (2 mM, Lonza, Basle, Switzerland), penicillin (100 U/mL, Lonza, Basle, Switzerland), and streptomycin (100 µg/mL, Lonza, Basle, Switzerland). Cells were maintained at 37 °C with 5% CO2 and 95% humidity.
9
Protein expression and gene silencing protocol
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Chemicals reagents used to prepare buffers and BCA Assay kit were purchased from Sigma Aldrich, UK and Fisher Scientific, UK. Buffers used include RIPA buffer, Phosphate-buffered saline (PBS), Tris-buffered saline (TBS), Blocking Buffer, Cell lysis buffer, elution buffer, SDS sample buffer, and ECL detection reagent [23] . PolyPlus INTERFERin was purchased from Source Bioscience, UK. Pre-designed siRNA, Opti-MEM, Power SYBR Green, RNa to cDNA kit, and gel casting materials were purchased from Life Technologies, UK.
Antibodies, Protein A/G agarose gel beads, and protein ladders were obtained from Santa Cruz Biotechnology, UK and Cell Signalling Technologies, UK. DMEM culture medium and other cell culture materials were purchased from Lonza, UK. PCR primers were designed with Primer3 Plus Bioinformatics Softwaew and NCBI BLAST, and purchased from Eurofins Genomics. The cells were incubated at 37ºC, 5% CO2 with Opti-MEM for 24 hours for efficient gene silencing.
Antibodies, Protein A/G agarose gel beads, and protein ladders were obtained from Santa Cruz Biotechnology, UK and Cell Signalling Technologies, UK. DMEM culture medium and other cell culture materials were purchased from Lonza, UK. PCR primers were designed with Primer3 Plus Bioinformatics Softwaew and NCBI BLAST, and purchased from Eurofins Genomics. The cells were incubated at 37ºC, 5% CO2 with Opti-MEM for 24 hours for efficient gene silencing.
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