MF patients’ or normal donors’ BM LDCs were cultured in conditions favoring differentiation to either fibrocytes or MSCs, as previously described [6 (link), 21 (link), 52 (link)]. To differentiate LDCs to fibrocytes BM LDCs were cultured in StemSpan serum-free medium (Stemcell Technologies, Vancouver, BC, Canada) supplemented with modified eagle medium (MEM), non-essential amino acid, insulin-transferrin-selenium, HEPES, glutamine, sodium pyruvate, penicillin, and streptomycin solutions (Sigma-Aldrich). To grow MSCs, BM LDCs were cultured in α-MEM supplemented with 20% FBS (Invitrogen, Waltham, MA, USA). All LDC-derived cultures were grown in Nunc Lab-Tek II CC2 chamber slides (Thermo Scientific, Waltham, MA, USA) at 37 °C in humidified atmosphere supplemented with 5% CO2.
Insulin transferrin selenium
Manufactured by Merck Group
Sourced in United States, United Kingdom, China, France, Ireland
Insulin-transferrin-selenium is a nutritional supplement used in cell culture media to promote cell growth and proliferation. It provides a combination of insulin, transferrin, and selenium, which are essential components for various cellular processes. This product serves as a defined and serum-free supplement to support the in vitro cultivation of a wide range of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (28 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Bovine serum albumin (bsa), by Merck Group (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Ascorbic acid, by Merck Group (6 mentions)
Penicillin streptomycin, by Merck Group (5 mentions)
L ascorbic acid 2 phosphate, by Merck Group (5 mentions)
L ascorbic acid, by Merck Group (5 mentions)
β glycerophosphate, by Merck Group (5 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Merck Group (4 mentions)
Dmem f12 medium, by Merck Group (4 mentions)
Ascorbate 2 phosphate, by Merck Group (4 mentions)
Linoleic acid, by Merck Group (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Evos microscope, by Thermo Fisher Scientific (4 mentions)
L proline, by Merck Group (3 mentions)
Insulin, by Merck Group (3 mentions)
84 protocols using insulin transferrin selenium
1
Differentiation of Bone Marrow Cells
2
Multilineage Differentiation of ADSCs
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For osteogenic and adipogenic differentiation, ADSCs were seeded on 6-well plates at the density of 1×105 cells/well. Osteogenic induction was performed using differentiation media consisting of DMEM-LG supplemented with 10% FBS, 50 mg/ml of ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone (all Sigma-Aldrich; Merck KGaA). The media was changed every 3 days until day 21.
Adipogenic studies were performed by culturing the cells in differentiation media containing 1 mM dexamethasone, 50 mM 3-isobutyl-1-methylxanthine and 10 mg/ml insulin (all Sigma-Aldrich; Merck KGaA).
For chondrogenesis, 1×105 cells were centrifuged at 500 × g in a polypropylene tube (15 ml; BD Biosciences) for 10 min at 4°C. Aggregates were incubated in chondrogenic media consisting of DMEM supplemented with 10% FBS, 1% insulin-transferrin-selenium, 1 mM sodium pyruvate and 50 mM L-proline (all Sigma-Aldrich; Merck KGaA).
After the differentiation processes were complete, cells were fixed in 4% paraformaldehyde for 30 min at 37°C and stained with Alizarin Red (10 min at 37°C), Oil Red O (10 min at 37°C) and Toluidine Blue (30 min at 37°C; all Beyotime Institute of Biotechnology), respectively. Then, cells were observed with a light microscope (Optiphot; Nikon Corporation) at a high-power magnification of ×100.
Adipogenic studies were performed by culturing the cells in differentiation media containing 1 mM dexamethasone, 50 mM 3-isobutyl-1-methylxanthine and 10 mg/ml insulin (all Sigma-Aldrich; Merck KGaA).
For chondrogenesis, 1×105 cells were centrifuged at 500 × g in a polypropylene tube (15 ml; BD Biosciences) for 10 min at 4°C. Aggregates were incubated in chondrogenic media consisting of DMEM supplemented with 10% FBS, 1% insulin-transferrin-selenium, 1 mM sodium pyruvate and 50 mM L-proline (all Sigma-Aldrich; Merck KGaA).
After the differentiation processes were complete, cells were fixed in 4% paraformaldehyde for 30 min at 37°C and stained with Alizarin Red (10 min at 37°C), Oil Red O (10 min at 37°C) and Toluidine Blue (30 min at 37°C; all Beyotime Institute of Biotechnology), respectively. Then, cells were observed with a light microscope (Optiphot; Nikon Corporation) at a high-power magnification of ×100.
3
3D Alveolar Epithelial Cell Culture
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CD31–CD45–EPCAM+HTII-280+ alveolar epithelial cells were resuspended in CK+DCI medium, 3D medium (DMEM/F12 supplemented with 10% FBS, penicillin/streptomycin 1,000 U/mL, 1 mM HEPES, 1 mM l -glutamate, and insulin/transferrin/selenium from Sigma), or SAGM as shown at a density of 5 × 103 cells/50 μL and then mixed with 3D Matrigel (Corning) containing MRC5 fibroblasts (ATCC CCL-171) at a density of 50 × 103 cells/50 μL. A total of 100 μL of the suspension was seeded on a 0.4 mm–pore cell culture insert in a 24-well supported format (Corning). After polymerization of Matrigel, 500 μL of CK+DCI medium was added to the bottom chamber. Medium was supplemented with 10 μM Y-27632 for the first 48 hours. Medium was changed every other day. Cultures were maintained at 37°C in a humidified incubator (5% CO2).
4
Efficient Generation of β-like Cells
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We used two steps protocol to get the β-like cells. The RPMI H-DMEM (Gibco) was used as base medium, added 10% FBS (Gibco), 2 mM glutamine (Invitrogen), 1 mM β-mercaptoethanol (Sigma), 10 mM nicotinamide (Sigma), 0.2% B27 (STEMCELL Technologies, Inc.), 1 mM sodium pyruvate (Invitrogen), 0.2% Insulin Transferrin Selenium (Sigma) and 25 ng/mL Activin A (Sigma) as the first stage induction medium. After 3 days, we added 25 mM zinc acetate, 10 nM exendin 4 (Sigma) and 4 nM beta-cellulin (Sigma) to the first stage induction medium as second stage induction medium. Then, we seeded and cultured the cells to ultra-low attachment plates for another 7 days. The induction medium was changed every 2 days.
5
Podocyte Injury and Recovery Protocol
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Human podocytes were cultured as described previously [3 (link),30 (link),31 (link)]. In brief, these cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640)-based medium supplemented with 10% fetal bovine serum (FBS; Invitrogen), 2g/L of NaHCO3, insulin-transferrin-selenium (Sigma-Aldrich, USA, St. Louis, MO, USA), and 200 units/mL penicillin and streptomycin (Roche) [3 (link),30 (link)]. The human podocytes were grown on collagen-coated culture dishes at 33 °C and 5% CO2, and were differentiated by thermo switching to 37 °C and by removing insulin-transferrin-selenium from the media, as described previously [3 (link),30 (link),31 (link)]. Podocyte injuries were developed using adriamycin (ADR) (0.25 μg/mL) for 48 h in serum-free media, whereas control podocytes were incubated only in the serum-free media [9 (link)]. The ADR-injured and control podocytes were processed for RNA isolation. In a similar fashion, the drug-induced recovery was initiated by adding Pifithrin-α (PFT-α) or vehicle in serum-free medium along with ADR.
6
Stem Cell Culture Protocol
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DMEM/F12 medium, dexamethasone, fetal bovine serum (FBS), insulin/transferrin/selenium (ITS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO). β-Mercaptoethanol, fibroblast growth factor 2 (FGF2), N2, and B27 supplements were from Thermo Fisher (Waltham, MA). Chicken embryo extract was from Biological (Salem, MA). Epidermal growth factor (EGF) was from PeproTech (Rocky Hill, NJ). Leukemia inhibitory factor (LIF) was from Millipore (Burlington, MA). Oncostatin-M and platelet-derived growth factor bb (PDGFBB) were from ProSpec (East Brunswick, NJ). Smoothened agonist (SAG) was purchased from Cayman Chemical (Ann Arbor, MI). Anti-R-phycoerythrin (PE) Magnetic Particles and BD IMag™ Buffer (10×) were from BD Biosciences (Franklin Lakes, NJ). Midazolam (stock solution: 5 mg/ml in aqueous solution) was from Jiangsu Enhua Pharmaceutical (Xuzhou, China). Human LH was from MyBioSource (San Diego, CA). Detailed information for other materials can be found in Supplementary Table S1 Supplementary Table S2 Supplementary Table S3
7
Characterization of FGFR3 Mutant BCa Cells
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The BCa cell line 97-7 expressing endogenous mutant FGFR3S249C (12 (link),14 (link)) was provided by Professor Margaret Knowles at the Leeds Institute of Cancer and Pathology, Cancer Research UK Clinical Centre, St. James's University Hospital (Leeds, UK). The FGFR3 wild-type (FGFR3WT) BCa cell line 5637 and T24 were obtained from the Cell Bank of the Chinese Academy of Sciences. 97-7 (FGFR3S249C) cells were cultured in Hams F12 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% (v/v) FBS (Gibco; Thermo Fisher Scientific, Inc.), 1× Insulin-Transferrin-Selenium (Sigma-Aldrich; Merck KGaA), 1× non-essential amino acids (Gibco; Thermo Fisher Scientific, Inc.) and 1 µg/ml hydrocortisone (Sigma-Aldrich; Merck KGaA). 5637 (FGFR3WT) and T24 (FGFR3WT) cells were cultured in RPMI 1640 and McCoy's 5A medium (all Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% (v/v) FBS. All cell lines were cultured in a humidified incubator at 37°C with 5% CO2.
8
Ovarian Organ Culture Modeling
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Ovaries were dissected from PD4 mice and washed three times in Leibovitz’s-15 medium (Gibco) containing 10% fetal bovine serum plus 1% penicillin–streptomycin before being transferred to culture inserts (Millipore) in a 6-well culture plate (Costar) at 37℃ and 5% CO2. DMEM/F12 (Gibco) supplemented with 5% Insulin-Transferrin-Selenium (Sigma), 1 mg/ml BSA (Sigma), 1 mg/ml Albumax II (Gibco), 100 µM L-ascorbic (Sigma), and 1% penicillin–streptomycin was used as the culture medium. A drop of medium was placed to cover the top of the ovary to prevent drying, and the ovaries were cultured for 4 or 8 days with medium changed every 2 days. Ovaries were treated with control medium (1% DMSO), VCD (30 µM), VCD + MSC-CM (fivefold concentration), or VCD + Fib-CM (fivefold concentration). Appropriate concentrations of recombinant human HGF (100–800 ng/ml), G-CSF (100–800 ng/ml), BDNF (100–800 ng/ml), or HGF neutralizing antibody (0–1 ng/ml) were added directly to the culture medium.
9
Human Retinal Endothelial and Muller Cell Stimulation
10
Isolation and Culture of Retinal Cells
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Primary human RECs and Müller cells were obtained as described.15 (link) Retinal endothelial cells were cultured in gelatin-coated flasks containing DMEM plus 10% FBS (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), endothelial cell growth supplement from bovine pituitary (15 μg/mL; Sigma-Aldrich Corp., St Louis, MO, USA) and insulin/transferrin/selenium (Sigma-Aldrich Corp.). Cell identity was confirmed by incorporation of acetylated low-density lipoprotein (>90%). Primary human Müller cells were cultured in DMEM/F12 containing 20% FBS. Cultures were >95% pure for Müller cells (vimentin+, CRALBP+, and GFAP− by immunofluorescence). Human retinal cells were used between passages 3 to 6. The human Müller cell line MIO-M1 was a gift from Gloria Limb (University College London, London, UK). In coculture experiments, 8 × 104 RECs and 4 × 104 RMCs were cultured per well of 6-well plates.
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