Hek a

Manufactured by Thermo Fisher Scientific

The HEK-A is a laboratory instrument designed for the cultivation and analysis of human embryonic kidney (HEK) cells. It provides a controlled environment for the growth and study of these cells, which are commonly used in various research and development applications.

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6 protocols using hek a

Primary HUVEC Culture and Transfection

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Pooled Human umbilical vein ECs (HUVECs) were purchased from PromoCell and cultured in EGM-2 media (PromoCell Growth Medium, ready-to-use) for 2–5 passages. For experiments, glass-bottomed imaging dishes were exposed to deep UV light for 6 min and coated with Poly-D-Lysine (ThermoFisher) for a minimum of 20 min. Small interfering RNA (Thermo Fisher) was introduced into primary HUVEC using the Neon® transfection system (Thermo Fisher). Scramble and Rab27a siRNAs were purchased from (Thermo Fisher) and resuspended to a 10 μM stock concentration and used at 0.5 μM (see data supplement). Normal human lung fibroblasts (NHLFs, Lonza) and HEK-A (Thermo Fisher) were maintained in Dulbeccos Modified Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin antibiotics. Both NHLFs and HEKs were used up to 15 passages. All cells were maintained in a humidified incubator at 37°C and 5% CO₂.

Francis C.R, & Kushner E.J. (2021). Capturing membrane trafficking events during 3D angiogenic development in vitro. Microcirculation (New York, N.y. : 1994), 29(6-7), e12726.

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Endothelial Cell Culture and Secretion Assay

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Pooled Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell and cultured in proprietary media (PromoCell Growth Medium, ready-to-use) for 2-5 passages. For experiments glass-bottomed imaging dishes were exposed to deep UV light for 6 minutes and coated with Poly-D-Lysine (ThermoFisher) for a minimum of 20 minutes. Small interfering RNA (ThermoFisher) was introduced into primary HUVEC using the Neon® transfection system (ThermoFisher). Scramble, Slp2a, Rab27a, and Slp4a siRNAs were purchased from (ThermoFisher) and resuspended to a 10μM stock concentration and used at 0.5 μM. Normal human lung fibroblasts (NHLFs, Lonza) and HEK-A (ThermoFisher) were maintained in Dulbeccos Modified Medium (DMEM) supplemented with 10% fetal bovine serum and pen/strep antibiotics. Both NHLFs and HEKs were used up to 15 passages. All cells were maintained in a humidified incubator at 37°C and 5% CO₂.
Phorbol myristate acetate (PMA) or histamine (Sigma) was used to induce secretion of WPB components. To achieve this, cells were serum-starved for 6 hours and treated with a final concentration of 100ng/mL PMA or 100 μM histamine for 15 minutes. Cells were then washed with phosphate buffered saline (PBS) and fixed promptly in 4% paraformaldehyde (PFA). For Tie-2 inhibition, cells were treated with BAY-826 (TOCRIS) at a final concentration of 1.3 nM for 1-3 days during sprouting.

Francis C.R., Claflin S, & Kushner E.J. (2021). Synaptotagmin-like protein 2a regulates angiogenic lumen formation via Weibel-Palade body apical secretion of angiopoietin-2. Arteriosclerosis, thrombosis, and vascular biology, 41(6), 1972-1986.

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Quantifying STAT Phosphorylation in Cells

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IL-6 and Oncostatin M (OSM) induced STAT3 phosphorylation was assessed in the human erythroleukemia TF-1 cell line. Erythropoietin-induced STAT5 phosphorylation was assessed in the human UT-7 cell line. IL-2 and IL-15 induced STAT5 phosphorylation was assessed in activated human T-cells. Detection of phosphorylated STATs was accomplished with the SureFire pSTAT5 or pSTAT3 Assay kit (Perkin Elmer, Hopkinton, MA) per standard manufacturer’s protocol, with the exception of an overnight incubation following addition of donor beads before detection on the EnVision (Perkin Elmer, Hopkinton, MA). IFNγ induced STAT1 phosphorylation was assessed in the CD14+ monocyte population in human PBMC by flow cytometry. CD14 BV421 was purchased from BD Biosciences (San Jose, CA). STAT1 PE (pY705) was purchased from ThermoFisher Scientific (Waltham, MA). IL-4 and IL-13 induced STAT6 phosphorylation and IL-31 induced STAT3 phosphorylation were assessed in adult human epithelial keratinocytes (HEKa, ThermoFisher Scientific, Waltham, MA) by flow cytometry. STAT6 PE (pY641) and STAT3 PE (Y705) were purchased from BD Biosciences (San Jose, CA).

Parmentier J.M., Voss J., Graff C., Schwartz A., Argiriadi M., Friedman M., Camp H.S., Padley R.J., George J.S., Hyland D., Rosebraugh M., Wishart N., Olson L, & Long A.J. (2018). In vitro and in vivo characterization of the JAK1 selectivity of upadacitinib (ABT-494). BMC rheumatology, 2, 23.

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Culturing Adult Human Epithelial and Dermal Cells

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Adult HEKa (Thermo Fisher Scientific, #C0215C) and HDFa (Thermo Fisher Scientific, #C0135C) were cultured in EpiLife Basal Medium supplemented with S7 and gentamicin/amphotericin B and Medium 106 supplemented with low serum growth supplement (LSGS), gentamycin, respectively (Thermo Fisher Scientific). T25 flasks (Nunc)
were coated with a Coating Matrix kit (Thermo Fisher Scientific) for 0.5 hours at room temperature prior to HEKa culture. Cells were maintained in a humidified incubator (37°C, 5% CO 2 ) and passaged using tryspin/versene at 80% confluency. All cell lines were confirmed mycoplasma negative prior to use using MycoAlert (Promega). Both HEKa and HDFa were used between passage 3 (p 3) and p10 for all assays.

Powell L.C., Cullen J.K., Boyle G.M., De Ridder T., Yap P.Y., Xue W., Pierce C.J., Pritchard M.F., Menzies G.E., Abdulkarim M., Adams J.Y.M., Stokniene J., Francis L.W., Gumbleton M., Johns J., Hill K.E., Jones A.V., Parsons P.G., Reddell P, & Thomas D.W. (2022). Topical, immunomodulatory epoxy-tiglianes induce biofilm disruption and healing in acute and chronic skin wounds. Science translational medicine, 14(662).

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Keratinocyte Viability Assay Protocols

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Cell viability assays were conducted to assess the effect of test articles in the vehicles at two different concentrations, 10 and 1. Primary normal human epidermal keratinocytes, adult (HEKa) (Thermo Fisher; catalog number C0055C) were seeded using supplemented medium 154 (Life Technologies; catalog number M154500) at 2 × 10⁵ cells/well and allowed to settle for 1 h. Cells were then treated with the test articles/vehicles, negative controls (medium and 10 phosphate‐buffered saline [PBS]). At 20 h posttreatment, AlamarBlue Cell Viability Reagent (Life Technologies; catalog number DAL1025) was added to each well, and 4 h later, the cell viability was assayed from triplicate wells by reading fluorescence intensity of AlamarBlue reagent on a fluorescence plate reader according to the manufacturer's instructions.
The test articles and controls were prepared as follows: dilutions of 1:10 (10) or 1:100 (1) of the test articles were made directly in the well by adding either 20 or 2 µl of the test articles to an appropriate amount of medium to reach the final volume of 200 µl in the well. The 1:10 and 1:100 dilutions of vehicles and saline controls were prepared in a similar fashion.

Haapakorva E., Raunio H., von Wright A, & Harvima I. (2022). Pinoresinol stimulates keratinocyte proliferation and downregulates TNF‐α secretion in peripheral blood mononuclear cells: An experimental in vitro study. Health Science Reports, 6(1), e998.

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Endothelial cell culture and siRNA transfection

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Pooled Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell and cultured in EGM-2 media (PromoCell Growth Medium, ready-to-use) for 2-5 passages. For experiments glass-bottomed imaging dishes were exposed to deep UV light for 6 minutes and coated with Poly-D-Lysine (ThermoFisher) for a minimum of 20 minutes. Small interfering RNA (ThermoFisher) was introduced into primary HUVEC using the Neon® transfection system (ThermoFisher). Scramble and Rab27a siRNAs were purchased from (ThermoFisher) and resuspended to a 10µM stock concentration and used at 0.5 µM (see data supplement). Normal human lung fibroblasts (NHLFs, Lonza) and HEK-A (ThermoFisher) were maintained in Dulbeccos Modified Medium (DMEM) supplemented with 10% fetal bovine serum and pen/strep antibiotics. Both NHLFs and HEKs were used up to 15 passages. All cells were maintained in a humidified incubator at 37 o C and 5% CO 2 .

Francis C.R., & Kushner E.J. (2021). Capturing Membrane Trafficking Events During 3D Angiogenic Development in Vitro.

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