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.
Huvecs
Manufactured by Takara Bio
Sourced in Japan
HUVECs (Human Umbilical Vein Endothelial Cells) are primary endothelial cells derived from the umbilical vein of human placenta. They are used as an in vitro model for the study of endothelial cell biology and physiology.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Endothelial cell growth medium 2, by Takara Bio (2 mentions)
Hepes bss, by Lonza (2 mentions)
Antibiotic antimycotic stock solution, by Nacalai Tesque (2 mentions)
Endothelial cell growth medium kits, by Takara Bio (2 mentions)
Trypsin edta, by Lonza (2 mentions)
Trypsin edta solution, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Cytiva (2 mentions)
Penicillin streptomycin amphotericin b, by Fujifilm (1 mentions)
Reprostem medium, by ReproCELL (1 mentions)
Fgm cd bullet kit, by Takara Bio (1 mentions)
Hep3b cells, by Sumitomo Pharma (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Nacalai Tesque (1 mentions)
Fetal bovine serum (fbs), by Nacalai Tesque (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
10 protocols using huvecs
1
HUVEC CLIC2 Knockdown Protocol
2
Cell Culture Protocols for HEK293 and HUVEC
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HEK293 cells were maintained by DMEM (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Cytiva, Marlborough, MA, USA) and Antibiotic-Antimycotic Stock Solution (NACALAI TESQUE, Inc., Kyoto, Japan). For replating cells, Trypsin-EDTA solution (Merck, Darmstadt, Germany) was used. HUVECs were purchased from Kurabo industries (Osaka, Japan) and Takara. HUVECs were maintained in Endothelial Cell Basal Medium 2 supplemented with Endothelial Cell Growth Medium kits (C22211, C22111, Takara). For replating cells, the cells were washed once with Hepes-buffered saline (Hepes-BSS), trypsinized with Trypsin-EDTA (CC5012, Lonza, Basal, Switzerland) and neutralized with Hepes-BSS/10% FBS. The cells were centrifuged to eliminate Hepes-BSS/10% FBS and plated with the growth medium. The cells were used by passage 5.
3
Culturing HEK293 and HUVEC cells
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HEK293 cells were maintained by DMEM (Thermo Fisher) supplemented with 10% Fetal Bovine Serum (Cytiva, Marlborough, MA, USA) and Antibiotic-Antimycotic Stock Solution (NACALAI TESQUE, Inc., Kyoto, Japan). For replating cells, Trypsin-EDTA solution (Merck, Darmstadt, Germany) was used. HUVECs were purchased from Kurabo industries (Osaka, Japan) and Takara. HUVECs were maintained in Endothelial Cell Basal Medium 2 supplemented with Endothelial Cell Growth Medium kits (C22211, C22111, Takara). For replating cells, the cells were washed once with Hepes-buffered saline (Hepes-BSS), trypsinized with Trypsin-EDTA (CC5012, Lonza, Basal, Switzerland) and neutralized with Hepes-BSS/10% FBS. The cells were centrifuged to eliminate Hepes-BSS/10% FBS and plated with the growth medium. The cells were used by passage 5.
4
Culturing HUVECs and Mouse Primary SSCs
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HUVECs (TaKaRa Bio) were cultured using EGM2 (TaKaRa Bio) and penicillin/streptomycin/amphotericin B (Wako). Isolated mouse primary SSCs were cultured using the mouse MesenCult Expansion Kit with L-glutamine (Wako) and penicillin/streptomycin/amphotericin B (Wako).
5
Evaluating miR-520d-5p Effects In Vitro and In Vivo
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To determine the in vitro and in vivo effects of hsa-miR-520d-5p expression and to explore its safety for future systemic administration, we used three cell lines and lentiviral vectors. Human iPSCs (hiPSCs) (HPS0002) were provided by the RIKEN BioResource Center Cell Bank (Ibaraki, Japan), and both human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblast (NHDF) cells were provided by TAKARA BIO Inc. (Tokyo, Japan). To examine the effect of miR-520d-5p on normal cells in vitro and in vivo, we used a human fibroblast cell line (NHDF-Ad derived from adults) and HUVECs, cultured in fibroblast basal medium (FBM)-2 medium using the fibroblast growth medium chemically defined (FGM-CD) Bullet Kit and EBM-2 using EBM-2 SingleQuots, respectively (TAKARA BIO Inc.). The hiPSCs were cultured in ReproStem medium (ReproCell, Tokyo, Japan) with 5 ng/mL basic fibroblast growth factor (bFGF)-2. In addition, the human mesangial cell line 293FT (Invitrogen Japan K.K., Tokyo, Japan) was used for producing miR-520d-expressing lentivirus as previously reported [13 (link)]. The 293FT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 0.1 mM minimum essential medium (MEM) non-essential amino acids solution, 2 mM l -glutamine, and 1 % penicillin/streptomycin.
6
Cell Culture Conditions for Hepatic and Endothelial Cells
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Hep3B cells (DS Pharma Biomedical, Osaka, Japan) were cultured in Eagle’s minimal essential medium (MEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with MEM non-essential amino acid solution, 10% fetal bovine serum (FBS) (HyClone, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich). HepG2 and HLE cells (JCRB cell bank, Osaka, Japan) were cultured in Dulbecco’s modified Eagle medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Human umbilical vein endothelial cells (HUVECs) (Takara Bio Inc, Shiga, Japan) were cultured in EndoGRO™-VEGF Complete medium (Millipore, NJ, USA) in the absence of antibiotics. All cell lines were incubated at 37 °C and 5% CO2 in a humidified incubator.
7
Co-culture of Osteoblast and Endothelial Cells
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Osteoblast-like MC3T3-E1, MG-63 cells (European Collection of Cell Culture) and human umbilical vein endothelial cells (HUVECs, PromoCell, Heidelberg, Germany) were cultured in the present study. MC3T3-E1 cells were cultured in alpha-modified minimal essential medium (MEMα; Sigma-Aldrich, St. Louis, MO, USA) with 10% of fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). MG-63 cells were cultured in Eagle’s minimal essential medium (EMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% non-essential amino acids (Sigma-Aldrich), and 10% of FBS. HUVECs were cultured in Endothelial cell growth medium 2 (EGM2; Takara Bio Inc., Shiga, Japan). All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Under co-culture conditions, both MG-63 cells and HUVECs were seeded in each AFS set on a 24 well culture plate in EGM2 at an initial cell number of 2 × 105 cells and 8 × 105 cells, respectively. As a control, we seeded MG-63 cells at the same cell numbers and cultured in EMEM medium.
8
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).
9
Culturing Human Cells for Viral Studies
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Human umbilical vein endothelial cells (HUVECs) (cat# D10013, Takara, Shiga, Japan,) were cultured in collagen-coated dishes (Corning, AZ, USA) containing endothelial growth medium (EGM) (Promocell, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin solution, and 0.1% amphotericin B (Gibco, Grand Island, NY, USA). The human embryonic kidney cell line (HEK293T) (cat# CRL11268, ATCC, Manassas, VA, USA), used for the preparation of Ebola VLPs was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin solution. All cells were incubated at 37 °C in a humidified 5% CO2 environment and were kept at a low passage number (below 20) for the experiments to maintain cell morphology and biological properties.
10
Irradiation Effects on HUVEC Viability
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Human umbilical vein endothelial cells (HUVECs; Takara Bio, Shiga, Japan) were grown in endothelial cell basal medium 2 (C-22011; Takara Bio) supplemented with growth factors and fetal bovine serum (FBS). All cells were cultured at 378C under humidified conditions with 5% CO 2 as a general culture condition for 14 days. The medium was changed every other day, and cells with passage numbers of 6-7 were used for the experiments. X-ray irradiation experiments were performed at Nippon Medical School irradiation facility using a Hitachi MBR-1520R-3 device (150 kV, 20 mA, aluminum equivalent 0.5 mm, copper filter 0.1 mm, calculated dose rate 0.98 Gy/min). Cells were irradiated using 2, 6 and 10 Gy of X rays to evaluate cell viability, 6 and 10 Gy of X rays to evaluate apoptosis, and 6 Gy of X rays to evaluate the extent of DNA damage and changes in intracellular adenine nucleotide content. Immediately after irradiation, the HUVEC growth medium was replaced with a medium from which Hx was removed or a medium to which exogenous Hx was added.
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