Human OS cells (HOS, MG-63, N-methyl-N-nitro-N-nitrosoguanidine (MNNG)/HOS and 143B) and human umbilical vein endothelial cells (HUVEC) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were authenticated by DNA short tandem repeat profiling and experiments were conducted within 6 months of resuscitation. OS cells were maintained in DMEM medium containing 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA), while HUVECs were maintained in 200PRF medium (Invitrogen) supplemented with low serum growth supplement (Invitrogen). All cell cultures were maintained at 37°C with 5% CO2. 143B cells were serum starved overnight and treated with KN-93 (10 μM) and/or CBO-P11 (1μM) (Millipore) for 24 hours [14 (link)].
Low serum growth supplement
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Australia
Low Serum Growth Supplement is a laboratory product designed to support cell culture growth in reduced serum conditions. It provides essential nutrients and growth factors to maintain cell viability and proliferation when serum levels are limited.
Automatically generated - may contain errors
Lab products found in correlation
Medium 200, by Thermo Fisher Scientific (38 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (24 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (20 mentions)
Huvecs, by Thermo Fisher Scientific (16 mentions)
Penicillin, by Thermo Fisher Scientific (11 mentions)
Streptomycin, by Thermo Fisher Scientific (11 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (10 mentions)
M200 medium, by Thermo Fisher Scientific (9 mentions)
Human umbilical vein endothelial cells (huvec), by Thermo Fisher Scientific (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Medium 200prf, by Thermo Fisher Scientific (7 mentions)
Mda mb 231, by American Type Culture Collection (5 mentions)
Huvecs, by Lonza (5 mentions)
Gentamicin, by Thermo Fisher Scientific (5 mentions)
Huvec cells, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Trypsin edta, by Thermo Fisher Scientific (5 mentions)
Haecs, by Thermo Fisher Scientific (4 mentions)
Medium 106, by Thermo Fisher Scientific (4 mentions)
Amphotericin b, by Thermo Fisher Scientific (4 mentions)
140 protocols using low serum growth supplement
1
Characterization of Osteosarcoma Cell Lines
2
Isolation of Small Extracellular Vesicles from Endothelial Cells
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Primary human umbilical vein endothelial cells (HUVECs; passage 4–6: Lonza, NJ, USA) and immortalized mouse aortic endothelial cells (iMAECs) were used to isolate sEVs. HUVECs were cultured in Medium 200 (Invitrogen, Carlsbad, CA, USA) containing 5% fetal bovine serum (FBS; HyClone, Melbourne, VIC, Australia), 2% low-serum growth supplement (Invitrogen, Carlsbad, CA, USA), and 1% antibiotic/antimycotic solution (Corning Cellgro, Manassas, VA, USA). The iMAECs were cultured in Dulbecco’s Modified Eagles Medium (DMEM; HyClone, South Logan, UT, USA) containing 10% FBS, 25 ug/mL endothelial cell growth supplement (ECGS; BD Biosciences, Franklin Lakes, NJ, USA), and 1% antibiotic/antimycotic solution. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
3
Culturing Endothelial Cell Lines for Research
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The human umbilical vein endothelial cell line (HUVEC, acquired from Lonza) was maintained in basal medium 199 (Invitrogen) with 20% fetal bovine serum (FBS), heparin (25 µg/mL, Sigma) and endothelial cell growth supplement (ECGS) (50 µg/mL, Sigma). The human bone marrow endothelial cell line (HBMEC-60) [32] (link) was maintained in basal medium 200 (Invitrogen) with 20% FBS and a low-serum growth supplement (Invitrogen). The amphotropic Phenix packaging cell line, H29 was maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) with 10% FBS (HyClone), 100 units/mL penicillin, 100 µg/mL streptomycin (Invitrogen) and 1 µg/mL tetracycline (Sigma) in 5% CO2 at 37°C.
4
Cytokine and Autophagy Responses in Fibroblast-Eosinophil Co-culture
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Primary human dermal fibroblasts (Life Technologies, Carlsbad, CA, United States) were grown in Medium 106 supplemented with low serum growth supplement (Gibco Invitrogen Corp., Carlsbad, CA, United States). Eosinophils (3 × 105 cells) and confluent fibroblasts (1 × 105 cells) were co-cultured in RPMI 1640 medium containing 10% FBS with or without IL-37b (100 ng/mL) pre-treatment for 10 mins, followed by IL-31 and IL-33 (100 ng/mL) stimulation for 24 h prior to the determination of the expression of cytokines/chemokines and autophagy related proteins.
5
3D Co-Culture of HUVECs and Duct Cells
6
Culturing Human Cell Lines for Research
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Human umbilical vein endothelial cells (HUVECs; cat. no. CRL-1730™) and normal human dermal fibroblasts (NHDFs; cat. no. PCS-201-012™) were purchased from the American Type Culture Collection, while HaCaT epidermal keratinocytes were provided by Professor Tae Yoon Kim from the College of Medicine of The Catholic University of Korea. All the cell lines used in this study were tested for mycoplasma contamination and used in experiments between passage numbers 5 and 10. HaCaT cells were cultured in high-glucose DMEM (Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HUVECs were cultured in M200 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 20% FBS and 1% low-serum growth supplement (Invitrogen; Thermo Fisher Scientific, Inc.). NHDFs were cultured in fibroblast medium (ScienCell Research Laboratories, Inc.). Cultures were at all times incubated in a humidified atmosphere at 37°C and 5% CO2 unless stated otherwise.
7
In Vitro Cytotoxicity Evaluation
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The synthesized compounds were evaluated for their in vitro cytotoxic activities against a human colorectal cancer cell line (HT-29) and normal dermal fibroblasts (PCS-201-012), obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained routinely in McCoy’s 5A (HT-29) or Dulbecco’s Modified Eagle's medium (PCS-201-012), supplemented with 10% fetal bovine serum, sodium bicarbonate (1.5 g/L), penicillin (100 UI/mL), streptomycin (100 µg/mL), and low serum growth supplement (Invitrogen) for dermal fibroblasts, at 37 °C and 5% CO2.
8
HUVEC Tube Formation Assay
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HUVECs were cultured in M200 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 20% FBS and 1% low-serum growth supplement (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C and 5% CO2. Each well of the 96-well plates was coated with 50 µl of Matrigel matrix and incubated at 37°C for 30 min. HUVECs were suspended in serum-free medium containing 1X LGS, and 1×106 HUVECs were seeded in each well of the 96-well plates pretreated with Matrigel matrix with the vehicle (M200) or NED416 for 18 h. Tube formation was captured using an inverted microscope (magnification, ×40; Nikon Corporation).
9
Comprehensive Eicosanoid and VEGFR2 Inhibitor Protocol
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HKE2 was prepared by total synthesis as described (10 (link)). Other eicosanoids and the VEGFR2 inhibitor vatalanib hydrochloride were obtained from Cayman Chemical. The human phospho-Receptor Tyrosine Kinase array kit was from R&D Systems. Recombinant human PTP1B was from Abcam. Trypsin, HBSS, Rhodamine B isothiocyanate-dextran (MW 70 kDa), phorbol 12-myristate 13-acetate, and bovine serum albumin were from Sigma. Fetal bovine serum and growth factor–reduced matrigel were from Corning. Medium 200PRF and low serum growth supplement were purchased from Invitrogen. The selective inhibitors U0126 (ERK), LY294002 (PI3K), and cell lysis buffer were from Cell Signaling. VEGF-165 (VEGF165) was purchased from PeproTech. Ketoprofen was purchased from Zoetis. The MMP inhibitor GM6001 was a kind gift from Dr Barbara Fingleton, Vanderbilt University School of Medicine, Department of Pharmacology. Organic coconut MCT oil containing no trans, monounsaturated, or polyunsaturated fat was purchased from a local supermarket.
10
TNBC Cell Line MDA-MB-231 Characterization
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The human TNBC cell line MDA-MB-231 (ER-low, PR-low and HER2-low) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and was maintained in F12 MEM (NO.12400-024,Gibco, NY,USA) supplemented with 2 mM L-glutamine, 10% FBS, and penicillin/streptomycin. The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Human umbilical vein endothelial cells (HUVEC) (BCRC No.H-UV001) were obtained from the Taiwan Medical Cell and Microbial Resources, Food Industry Research Development Institute, Taiwan and were cultured in Medium 200 supplemented with low serum growth supplement (Invitrogen, Carlsbad, CA, USA). Cells at passages from three to ten were used for all experiments. Recombinant human BDNF (PeproTech, Rocky Hill, NJ, USA) used as a positive control and was purchased commercially. Inhibitors of ERK (PD98059, # P215; Sigma Aldrich Co., Saint Louis, USA), PI3K (LY-294,002, # L9908; Sigma Aldrich Co., Saint Louis, USA), and TrkB (GNF5837, # 4559; Tocris Bioscience Co., Bristol, UK)were commercially available. They were pre-treated 1 h before the administration of BDNF in cell cultures.
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