Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6. Experiments were repeated with endothelial cells from different lots. HUVECs and HCAECs were cultured in endothelial cell growth medium (Promocell; C-22010 and C-22020) or 2% (v/v) FBS endothelial cell basal medium (Promocell; C-22210 and C-22220) supplemented with 100 mg/mL penicillin and 100 IU/mL streptomycin. Human recombinant TNF-α protein was procured from R&D Systems (210-TA-20). Pharmacological inhibitor of β-catenin, iCRT-14, was acquired from Tocris Bioscience (4299). Cells were maintained in a humified atmosphere with 5% CO2 at 37°C.
C 12221
Manufactured by PromoCell
Sourced in Germany
The C-12221 is a lab equipment product. It is designed for laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Icrt14, by Bio-Techne (1 mentions)
Hcaec, by PromoCell (1 mentions)
C 22010, by PromoCell (1 mentions)
C 22210, by PromoCell (1 mentions)
C 22220, by PromoCell (1 mentions)
C 12203, by PromoCell (1 mentions)
C 22020, by PromoCell (1 mentions)
Huvecs, by PromoCell (1 mentions)
Pcs 100 020, by American Type Culture Collection (1 mentions)
Prism 8, by GraphPad (1 mentions)
Spss statistics 25, by IBM (1 mentions)
Cell strainer, by Thermo Fisher Scientific (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Crl 1458, by American Type Culture Collection (1 mentions)
Crl 1927, by American Type Culture Collection (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (1 mentions)
Nunc thermanox coverslips, by Thermo Fisher Scientific (1 mentions)
4 protocols using c 12221
1
Comparison of HUVEC and HCAEC Responses
2
Detecting Physiological Differences in Cell Cultures
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Four experiments were needed to detect a physiologically relevant difference of 25% between control and intervention, given a standard deviation of 10%, a power of 80%, and an α of 0.05. Data are presented as mean ± standard deviation (SD). The distribution of the data was tested using the Shapiro-Wilk test. Normal distributed data were tested by one-way ANOVA with Bonferroni post hoc testing or by a Student’s t test. Non-normally distributed data were tested with Kruskal-Wallis and Mann-Whitney U test with Bonferroni correction. In each experiment, we attempted to avoid experimental bias by single-well use by pooling two wells with cells. The number of experiments mentioned in the figures refers to the number of technical replicates with cells from two donors (PCS-100-020 from ATCC, LOT# 59885589 and C-12221 from PromoCell, LOT# 425Z0191.1). Graphs were created in Graphpad Prism 8 and statistical analysis was performed using IBM SPSS Statistics 25. The cut-off values for statistical significance were indicated in the figures by *, **, and *** for p < 0.05, p < 0.01, and p < 0.001, respectively.
3
Cardioprotective Effects of Conditioned Media
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L6 rat skeletal myoblasts (CRL-1458; ATCC, Manassas, USA), Huh7 human hepatoma cells (JCRB0403; JCRB Cell Bank, Osaka, Japan), MES13 mouse renal mesangial cells (CRL-1927; ATCC), primary human coronary artery endothelial cells (C-12221; PromoCell, Heidelberg, Germany), primary rat aortic smooth muscle cells, and primary rat hepatocytes were plated each on a 100 mm dish. These cells were incubated at 37 °C in DMEM containing 1% glucose (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS, antibiotics (Gibco, Waltham, USA), and non-essential amino acids (Gibco) to become confluent, and serum-starved for 24 hrs. Each conditioned medium was collected and filtered by a cell strainer (Thermo Fischer Scientific, Waltham, USA). In parallel, primary neonatal rat cardiomyocytes were serum-starved for 24 hrs and subjected to 10−6 M doxorubicin (Sigma-Aldrich, St. Louis, USA) for 12 hours or 1% hypoxia by AnaeroPack Kenki (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) for 9 hrs, with or without administration of the above conditioned medium. After stimulation, Akt and caspase-3 activation was analyzed in cell lysates of the cardiomyocytes by Western blot.
4
Influence of RV on Endothelial Cell Viability and NO Production
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To examine the influence of RV on endothelial cell viability and production of NO, human coronary artery endothelial cells (HCAEC, Promo cell, C-12221) were grown as a monolayer at 37°C under 5% CO2 atmosphere in EC growth medium MV (Promo cell, C-22020) supplemented with 5% foetal calf serum, 4 LmL -1 growth medium, 0.5 ng/mL -1 vascular endothelial growth factor, 10 ngmL -1 epidermal growth factor and 90 μgmL -1 heparin.
Before use, cells were washed three times in phosphate buffered saline (PBS) and then incubated with 0.1% trypsin for three minutes to detach them from the flask surface, centrifuged for 3 min at 220 g, and re-suspended in growth medium for seeding. Cells were seeded onto Nunc Thermanox coverslips (10,000 cells/coverslip; 13 mm diameter) (Thermo Fisher Scientific, USA) and allowed to proliferate for 24 hours (approximately 90% confluency). Half of the coverslips were kept in the well plate to perform the ACh assay while the other overslips were transferred into a millifluidic system made up of a bioreactor chamber (Kirkstall, UK) attached with a flow pump system in which increased flow was introduced [41] as indicated in the section below. All reagents were obtained from Sigma-Aldrich (Poole, UK), unless otherwise stated.
Before use, cells were washed three times in phosphate buffered saline (PBS) and then incubated with 0.1% trypsin for three minutes to detach them from the flask surface, centrifuged for 3 min at 220 g, and re-suspended in growth medium for seeding. Cells were seeded onto Nunc Thermanox coverslips (10,000 cells/coverslip; 13 mm diameter) (Thermo Fisher Scientific, USA) and allowed to proliferate for 24 hours (approximately 90% confluency). Half of the coverslips were kept in the well plate to perform the ACh assay while the other overslips were transferred into a millifluidic system made up of a bioreactor chamber (Kirkstall, UK) attached with a flow pump system in which increased flow was introduced [41] as indicated in the section below. All reagents were obtained from Sigma-Aldrich (Poole, UK), unless otherwise stated.
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