Caco-2 cells (ATCC, Rockville, MD) were cultured using DMEM/F12 (Gibco-BRL, Gaithersburg, MD, USA) containing 10% FBS and 1% antibiotic and antimycotic solution in a humidified atmosphere of 5% CO2/95% air at 37 °C. Human umbilical vein endothelial cells (HUVEC) pooled cells were purchased from Promo Cell (Heidelberg, Germany) and cultured in endothelial cell growth medium 2 (ready-to-use; Takara-bio) for passage and expansion. For experiments, cells were seeded in 12-, 96-well culture plate or inserts until confluence and exposed to different treatments for the indicated times.
Endothelial cell growth medium 2
Manufactured by Takara Bio
Sourced in Germany, Japan
Endothelial Cell Growth Medium 2 is a specialized cell culture medium designed to support the growth and maintenance of endothelial cells. It provides the necessary nutrients and growth factors to sustain endothelial cell proliferation and function.
Automatically generated - may contain errors
Lab products found in correlation
Huvecs, by Takara Bio (2 mentions)
Caco 2 cell, by American Type Culture Collection (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Anti cd31 and anti cd102 antibodies, by BD (1 mentions)
Collagenase dispase solution, by Roche (1 mentions)
Dynabeads, by Takara Bio (1 mentions)
Rat anti mouse cd31 antibody, by BD (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (1 mentions)
Dnase 1, by Roche (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Gist t1, by Cosmo Bio (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Microscope camera system, by Nikon (1 mentions)
Cycleavepcr mycoplasma detection kit, by Takara Bio (1 mentions)
Hdmec, by Takara Bio (1 mentions)
Human umbilical vein endothelial cells (huvec), by Takara Bio (1 mentions)
5 protocols using endothelial cell growth medium 2
1
Culturing Caco-2 and HUVEC Cells
2
HUVEC CLIC2 Knockdown Protocol
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Human umbilical vein endothelial cells (HUVECs; Takara Bio) were maintained in endothelial cell growth medium 2 (Takara Bio)34 Only 2nd and 3rd passaged HUVECs were used for the following experiments. For the knockdown of CLIC2 expression, HUVECs were seeded at 50,000 cells/cm2 and allowed to grow to 90%–100% confluency. Knockdown was performed using RNA interference as described previously41 The siRNA CLIC2 gene-targeting duplexes used were as follows: 5′-GCUAUAUUUGUGAUCAGAUTT-3′ and 5′-AUCUGAUCACAAAUAUAGCTT-3′ (Sigma-Aldrich). HUVECs were transfected with 20 nM of the labeled siRNA duplexes, SilenceMag siRNA delivery reagents (OZ Bioscience) on top of a magnetic plate (OZ Bioscience). The cells were incubated for 24 h with siRNA, then the culture medium was exchanged and the cells were maintained in fresh culture medium for 48 h. Western blotting was employed to confirm decreased CLIC2 protein expression. As a control, a siRNA duplex with an irrelevant sequence (irr-siRNA; 5′-GCGCGCUUUGUAGGAUUCGTT-3′ and 5′-CGAAUCCUACAAAGCGCGCTT-3′, Dharmacon Research, Pittsburgh, PA) was used.
3
Isolation of Mouse Lung Endothelial Cells
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Lung tissue was excised from an anesthetized mouse (1-week-old), minced, and then incubated in Dulbecco’s Modified Eagle’s Medium (DMEM; Nacalai Tesque, Kyoto, Japan) containing 1 mg/ml collagenase/dispase solution (Roche, Basel, Switzerland) and 5 U/ml DNase I (Roche) for 45 min at 37 °C. The digested pieces were further minced by passing them through a 20-gauge needle and then filtered with a 70-μm cell strainer (BD Biosciences, San Jose, CA, USA). The filtrate was centrifuged for 5 min at 400×g at 20 °C, and the resulting cell pellet was suspended in PBS containing 0.1% BSA. The cells were incubated with Dynabeads (Invitrogen, Carlsbad, CA, USA) precoated with rat anti-mouse CD31 antibody (BD Biosciences) for 30 min at room temperature. ECs bound to the Dynabeads were collected with a magnet, washed using PBS with 0.1% BSA, and then cultured in a 60-mm dish coated with 2% gelatin in Endothelial Cell Growth Medium 2 (Takara). The purity of isolated ECs was > 95%, which was confirmed by immunofluorescence microscopy using anti-CD31 and anti-CD102 antibodies (BD Biosciences).
4
GIST-T1 Cell Line Maintenance Protocol
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A human GIST cell line, GIST-T1, which is derived from a metastatic pleural tumor from gastric GIST in a Japanese woman, was purchased from Cosmo Bio. It harbors a heterozygous c-kit gene mutation at exon 11 (an in-frame deletion of 19 amino acids from Val560 to Tyr578). The GIST-T1 cell line was maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum (FBS) (BioWest), 100 U/ml of penicillin G and 100 µg/ml of streptomycin (Invitrogen; Thermo Fisher Scientific) at 37°C in 5% CO2. Human umbilical vein endothelial cells (HUVECs) (Takara) were grown in Endothelial Cell Growth Medium 2 (Takara).
5
Culturing Endothelial and Angiosarcoma Cells
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HUVEC (Takara Bio, Kusatsu, Japan; C-12203), HDMEC (Takara Bio; C-12212), HDBEC (Takara Bio; C-12211), and HAMON human angiosarcoma cells44 (link),45 (link) (kindly provided by Riichiro Abe, Division of Dermatology, Niigata University) were cultured in Endothelial Cell Growth Medium 2 (Takara Bio; C-22111), containing 2% fetal calf serum, 5 ng/mL human epidermal growth factor, 0.2 μg/mL hydrocortisone, 22.5 μg/mL heparin, 20 ng/mL R3 insulin-like growth factor-1, 1 μg/mL ascorbic acid, 10 ng/mL human basic fibroblast growth factor, and 0.5 ng/mL VEGF. ISO-HAS-B human angiosarcoma cells (Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University) were cultured in DMEM (Sigma Aldrich; D6429) supplemented with 10% FBS. Cells were passaged at confluence using a Detach Kit (Takara Bio; C-41210) according to the manufacturer’s instructions, and the medium was changed every 2–3 days. The CycleavePCR Mycoplasma Detection Kit (Takara Bio; CY232) was used to test for mycoplasma contamination, and cells were confirmed to be mycoplasma free. Cell morphology was observed under a microscope and pictures were captured using a microscope camera system (Nikon).
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