HEK293 were grown in DMEM containing glucose 1 g/l, pyruvate and GlutaMAX and supplemented with 10% fetal bovine serum, 1% non-essential amino acids, HEPES 10 mM and 1% penicillin and streptomycin (all from Life Technologies) at 37°C in a water humidified 5% CO2 incubator. Cells were sub-cultured twice a week at 90% confluence. HUVEC (passages 1 to 5) were obtained from Gibco and cultivated as described in Ruiz et al. (2017) (link). Briefly, cells were grown in M200 medium (Gibco) containing the Low Serum Growth Supplement (Gibco) and 1% penicillin and streptomycin. Cells were sub-cultured twice a week at 90% confluence, and cultivated on treated plastic flask and multi-dish plates (Nunc). For FRAP and Laurdan dye experiments, HEK293 or HUVEC were seeded in glass bottom dishes (Ibidi) pre-coated with 0.1% porcine gelatin (Sigma). The HEK293 cell line was authenticated by genotyping and confirmed to be free of mycoplasma.
Hepes 10mm
Manufactured by Thermo Fisher Scientific
Sourced in United States
HEPES 10mM is a buffering agent commonly used in cell culture media and biochemical applications to maintain a stable pH environment. It is a 10 millimolar (mM) solution of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in water.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Porcine gelatin, by Merck Group (2 mentions)
Multi dish plates, by Thermo Fisher Scientific (2 mentions)
Glutamax 1, by Thermo Fisher Scientific (2 mentions)
Laminin 1, by Merck Group (2 mentions)
Pluronic acid f127 2, by Merck Group (2 mentions)
M200 medium, by Thermo Fisher Scientific (1 mentions)
Low serum growth supplement, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by Thermo Fisher Scientific (1 mentions)
Glass bottom dishes, by Ibidi (1 mentions)
Human umbilical vein endothelial cells (huvec), by Ibidi (1 mentions)
Tryple express reagent, by Thermo Fisher Scientific (1 mentions)
Accutase, by GE Healthcare (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Rock inhibitor y 27632, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Dispase 2, by Roche (1 mentions)
Collagenase xi, by Merck Group (1 mentions)
9 protocols using hepes 10mm
1
Cell Culture Protocol for HEK293 and HUVEC
2
Culturing HEK293 and HepG2 Cell Lines
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HEK293 and HepG2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and grown in DMEM containing glucose 1 g/l, pyruvate and GlutaMAX and supplemented with 10% fetal bovine serum, 1% non-essential amino acids, HEPES 10 mM and 1% penicillin and streptomycin (all from Life Technologies, Carlsbad, USA) at 37 °C in a water-humidified 5% CO2 incubator. Cells were subcultured twice a week at 90% confluence. TrypLE Express reagent (Gibco, Carlsbad, USA) was used to detach HEK293 and Accutase (GE Healthcare, Chicago, USA) was used to detach HepG2 cells. All cell types were cultivated on treated plastic flasks and multidish plates (Nunc, Roskilde, Denmark) and for HepG2 coated with 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, USA).
3
Establishment of Human CRC Organoids
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Approximately 1 cm3 of tumor was cut into small pieces and washed with basal Advanced Dulbecco’s modified Eagle medium (DMEM)/F12 (Gibco, Eggenstein-Leopoldshafen, Germany) supplemented with Primocin 1× (Invivogen, Toulouse, France), Glutamax 1× (Thermo Fisher, Waltham, MA, USA), and HEPES 10 mM (Thermo Fisher) until the supernatant was clear. The tissue was digested with 1 mg/mL Dispase II (Roche, Basel, Switzerland) and 0.1 mg/mL Collagenase XI (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C. Regular gentle inversion every 5 min and frequent observation of tumor disaggregation were performed until small tumor patches became visible. Around 150 tumor patches were picked under a stereomicroscope, centrifuged (5 min, 200× g), resuspended in 20 µL Matrigel (Corning, Corning, NY, USA), and overlaid with a human colon medium [25 (link)] supplemented with 10 µM ROCK inhibitor Y-27632 (Sigma-Aldrich). The established human CRC organoids were cultured as described earlier [25 (link)] by passaging once a week with a split ratio of 1:4.
4
Murine Small Intestinal Organoid Culture
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All lines were cultured in drops of Cultrex PathClear Reduced Growth Factor Basement Membrane Extract Type 2 (Amsbio, 3533-005-02) in murine small intestinal organoids medium containing advanced DMEM/F12 medium (adDMEM/F12; Thermo Fisher Scientific, cat. no. 12634-010), GlutaMAX 1% (Thermo Fisher Scientific, cat. no. 35050-068), HEPES 10mM (Thermo Fisher Scientific, cat. no. 15630-056), 1x Penicillin/streptomycin (10,000 U/ml; Thermo Fisher Scientific, cat. no. 15140-122). B27 2% (Thermo Fisher Scientific, cat. no. 17504-044), N-acetylcysteine 1.25 mM (Sigma-Aldrich, cat. no. A9165), mEGF 50ng/ml (Peprotech, cat. no. 315-09), Noggin and R-spondin1 both 10% (conditioned medium prepared in house).
5
Differentiation and Stimulation of Murine Dendritic Cells and Macrophages
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Bone marrow derived dendritic cells (BMDCs) and bone marrow derived macrophages (BMDMs) were differentiated from bone marrow cells, obtained from the mice detailed above, as previously described [29 (link)]. In brief, cells were grown in complete RPMI 1640 with GlutaMAX (Gibco) with 10% FCS (Biowest), 1mM sodium pyruvate, HEPES 10mM, MEM non-essential amino acids, 2Mercaptoethanol 0,05mM and gentamicin 0,02mg/ml (all from Gibco). BMDCS and BMDMs were differentiated with GM-CSF from J558L cell line supernatants [30 (link)] or M-CSF from L929 fibroblast cell line supernatants, respectively. At day 8 and 7, respectively, BMDCs were 80% CD11c+CD11b+ and BMDMs were 95% CD11c-CD11b+. Total cells were harvested and cultured in 6-well plates (3x106/well) and stimulated for 6 h with killed or live L. major promastigotes at a parasite/cell ratio of 5:1, or 250 ng/ml of CpG 1826 or 100ng/ml of conventional LPS (E. coli, O111:B4, Sigma). Cells were harvested at 6h after stimulation for RNA extraction. At 24h, cell-free supernatants were harvested for cytokine determination by specific ELISA.
6
ECM Micropatterning for Organoid Cultures
7
ECM Micropatterning for Organoid Cultures
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Organoid monolayers were confined in large circular ECM micropatterns (>900 μm). For this, PAA gels were functionalized with 2mg/mL of Sulpho-SANPAH (Cultek) irradiated for 7.5 minutes with UV light (365nm). Gels were then washed twice with HEPES 10mM (Gibco). For ECM micropatterning, Polydimethylsiloxane (PDMS) stencils with circular openings were used25 (link). PDMS stencils were incubated with Pluronic acid F127 2% in PBS (Sigma-Aldrich) for 1 hour. Then, they were washed twice with PBS and allowed to dry at RT for 20 minutes. Stencils were carefully placed on top of the functionalized PAA gels. A solution of 250 μg/mL rat tail type I Collagen (First Link UK) and 100 μg/mL Laminin I (Sigma-Aldrich) dissolved in PBS was added on top of the PDMS stencils and incubated overnight at 4°C. Finally, the ECM solution was aspirated, the gels were washed twice with PBS and the PDMS stencils were carefully removed.
8
Generating Monocyte-Derived Dendritic Cells
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Monocyte-derived Dendritic cells (MoDCs) were generated by culturing CD14+ monocytes (106/mL) in-vitro for 7 days in complete medium: RPMI 1640 GlutaMAX (cat no. 61870, Gibco) supplemented with 10% heat inactivated serum (cat no. CVSVF00-01, Eurobio), 1% PS (cat no. 15140, Gibco), HEPES 10 mM (cat no. 15630, Gibco), in the presence of 50 ng/mL interleukin-4 (IL-4, cat no. 130-093-922, Miltenyi) and 100 ng/mL GM-CSF (cat no. 130-093-866, Miltenyi) (26 (link)).
After 7 days, and prior to co-culture, MoDCs were tested for differentiation and maturation status by measuring CD1a, CD83, CD86, Tim-3, PD-L1 and HLA-DR expression by flow cytometry.
After 7 days, and prior to co-culture, MoDCs were tested for differentiation and maturation status by measuring CD1a, CD83, CD86, Tim-3, PD-L1 and HLA-DR expression by flow cytometry.
9
PBMC Isolation and Stimulation for Cytokine Profiling
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PBMCs were obtained from the heparinized blood samples of 20 SSc patients. The PBMCs were isolated using the standard Ficoll-Hypaque density-gradient centrifugation (GE Healthcare Biosciences, Pittsburgh, PA, USA) method. PBMCs (1 × 106 cells/mL) were cultured in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), HEPES 10 mM (Gibco, Carlsbad, CA, USA), and penicillin (10.000 U/mL)/streptomycin (10.000 μg/mL) (Gibco, Carlsbad, CA, USA). Cells were stimulated with anti-CD3/CD28 (eBioscience, San Diego, CA, USA), and after 48 h culture supernatant was collected for quantification of cytokines.
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