Haoec

Manufactured by PromoCell

Sourced in Germany, United Kingdom

HAoECs are primary human aortic endothelial cells. They are a type of cell culture model derived from the aorta, which is the main artery that carries blood from the heart to the rest of the body.

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Primary HAoECs (2 citations) Human Aortic Endothelial Cell (HAoEC) (2 citations)

36 protocols using haoec

Culture of Human Aortic Endothelial Cells

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Human aortic endothelial cells (HAoEC) were purchased from PromoCell, Germany. HAoEC were cultured strictly following the manufacturer's recommendations using Endothelial Cell Growth Media MV2 and the PromoCell Split Kit. HAoEC were used up to passage 9. The media was supplemented with the supplement mix provided by the manufacturer, which contains 4% FBS and 2.5 μg/ml free cholesterol.

Baumer Y., Dey A.K., Gutierrez-Huerta C.A., Khalil N.O., Sekine Y., Sanda G.E., Zhuang J., Saxena A., Stempinski E., Elnabawi Y.A., Dagur P.K., Ng Q., Teague H.L., Keel A., Rodante J.A., Boisvert W.A., Tsoi L.C., Gudjonsson J.E., Bleck C.K., Chen M.Y., Bluemke D.A., Gelfand J.M., Schwartz D.M., Kruth H.S., Powell-Wiley T.M., Playford M.P, & Mehta N.N. (2020). Hyperlipidaemia and IFNgamma/TNFalpha Synergism are associated with cholesterol crystal formation in Endothelial cells partly through modulation of Lysosomal pH and Cholesterol homeostasis. EBioMedicine, 59, 102876.

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Isolation and Culture of Primary Human Vascular Cells

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Primary human aortic endothelial cells (HAoEC, Cat#: C-12272), primary human umbilical vein endothelial cells (HUVEC, Cat#: C-12253), primary human skin fibroblasts (HSF, Cat#: C-12352) as well as primary human aortic smooth muscle cells (VSMC, Cat#: C-12532) (all PromoCell, Germany) were cultured according to manufacturer’s recommendation using the corresponding media as well as the split kit (all, PromoCell, Germany). Cells were used up to passage 8. Human monocyte-derived macrophages (HMDM) were isolated from whole blood from consenting healthy volunteers by Histopaque (Sigma-Aldrich) density gradient centrifugation. Informed consent was obtained from all donors and IRB approval was received under protocol CHS# 19345 prior to experiments involving HMDM. Cells from the buffy coat were plated with hematopoietic X-VIVO 15 medium (purchased from Lonza, Switzerland) supplemented with 20% heat inactivated human serum for 1 hour before non-adherent cells were washed away. Adherent cells were maintained for 5 days on glass slide chambers before being used in experiments.

Baumer Y., McCurdy S., Weatherby T.M., Mehta N.N., Halbherr S., Halbherr P., Yamazaki N, & Boisvert W.A. (2017). Hyperlipidemia-induced cholesterol crystal production by endothelial cells promotes atherogenesis. Nature Communications, 8, 1129.

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Quantifying MAC Adhesion to Activated Endothelium

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MAC adhesion to a TNFα-activated endothelial monolayer was measured. MACs were detached and labelled using Cell Tracker™ Green CMFDA (Molecular Probes, USA). Confluent monolayers of human aortic endothelial cells (HAoEC, Promocell, Germany) were treated for 6 hours with 10 ng/ml TNFα. We have previously demonstrated that this is sufficient to up-regulate expression of adhesion molecules in this cell type. MACs were allowed to adhere to the HAoECs for 1 hour, non-adherent cells removed, and the number of MACs quantified in random fields. Relative adhesion was expressed and the mean number of cells adherent to TNFα-activated HAoECs compared to resting endothelial cells.

Reynolds J.A., Haque S., Williamson K., Ray D.W., Alexander M.Y, & Bruce I.N. (2016). Vitamin D improves endothelial dysfunction and restores myeloid angiogenic cell function via reduced CXCL-10 expression in systemic lupus erythematosus. Scientific Reports, 6, 22341.

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Culturing and Transfecting Aortic Cells

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Human aortic endothelial cells (HAoEC, Promocell Inc., Heidelberg, German) were cultured in endothelial growth medium MV2 (C-22022, Promocell Inc., Heidelberg, Germany) with the addition of growth medium MV2 supplement mix (C-39226, Promocell Inc., Heidelberg, Germany), and human aortic smooth muscle cells (HAoSMC, Promocell Inc., Heidelberg, Germany) were cultured in vascular smooth muscle cell basal medium (M231500, Thermo Fisher Scientific Inc., Waltham, MA, USA) with the addition of smooth muscle growth supplement (S00725, Thermo Fisher Scientific Inc., Waltham, MA, USA). Cell transfection with siRNA duplexes was performed using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) following the manufacturer’s instruction.

Lin T.F., Chou C.L., Hsieh C.J., Wu Y.J., Chen Y.C., Wu T.W., Lu S.X., Juang Y.L, & Wang L.Y. (2022). Association of Common Variants in OLA1 Gene with Preclinical Atherosclerosis. International Journal of Molecular Sciences, 23(19), 11511.

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Isolation and Characterization of Primary Endothelial Cells

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Primary human dermal LEC isolated from adult skin and human aortic endothelial cells (HAoEC) were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated fetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit. Cells were maintained in a humidified incubator at 37°C and used till passage 7. Primary mouse lung LEC were isolated after digestion of lungs with collagenase type 2 (1 mg/mL) for 1 h at 37°C as we described previously ^{24 (link)}. LYVE-1-positive cells were separated and cultured.
A smart pool of siRNA for human CD47 (M-019505-01-0005) and a non-targeting control siRNA (D-001210-01-05) were purchased from Horizon Discovery (Cambridge, United Kingdom). Cells were transfected with siRNAs using the TransIT-TKO transfection reagent (Mirus Bio LLC, Madison, WI, USA) according to the manufacturer’s instructions. Cells were used for further experiments 48 h post-transfection and gene silencing was confirmed using qRT-PCR. Protein knockdown was validated by Western blot experiments.

, & Shrier I. (2001). Uncertainty about clinical equipoise. CMAJ: Canadian Medical Association Journal, 164(13), 1831.

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Measurement of Intracellular Nitric Oxide

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HAoEC (PromoCell) were cultured in DMEM without phenol red containing l-glutamine (584 mg/liter) and fetal bovine serum (10%) (Thermo Fisher Scientific). The cells were seeded in 12-well plates at a density of 2 × 10⁵ cells per well and allowed to grow for another 2 days, followed by serum fasting overnight and subsequent stimulation with ACh (10 μM) in the absence or presence of recombinant human IL-1β (10 ng/ml; Sigma-Aldrich) for 15 min. For measurement of intracellular NO, 4,5-diaminofluorescein diacetate (DAF-2DA; 10 μM) (Sigma-Aldrich) was added to the cells for the last 5 min in the dark (37°C, 5 min). Fluorescence images were captured. Alternatively, HAoEC were seeded in 96-well plates at a density of 0.25 × 10⁵ cells per well and grown for 2 days. DAF-2DA (10 μM) was added 5 min before harvesting, and the fluorescence intensity of the cells was measured using a spectrofluorometer with excitation wavelength at 495 nm and emission wavelength at 515 nm. The relative fluorescence intensity was normalized with protein concentration in cell lysate.

Gu P., Hui X., Zheng Q., Gao Y., Jin L., Jiang W., Zhou C., Liu T., Huang Y., Liu Q., Nie T., Wang Y., Wang Y., Zhao J, & Xu A. (2021). Mitochondrial uncoupling protein 1 antagonizes atherosclerosis by blocking NLRP3 inflammasome–dependent interleukin-1β production. Science Advances, 7(50), eabl4024.

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Bacterial Adherence to Endothelial Cells

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The bacterial strains used in this study are S. aureus NCTC 8325-4 and isogenic mutant lacking the fibronectin binding proteins, S. aureus 8325-4 ΔfnbpAB. Both isolates were grown in Brain Heart Isolation (BHI) broth to mid-exponential growth phase at 37 °C (optical density 600 nm = 1). Human Aortic Endothelial Cells (HAoEC) (PromoCell) were maintained in Endothelial Cell Media supplemented with basal medium, penicillin (200 U/mL) and streptomycin (200 µg/mL).

Nader D., Yousef F., Kavanagh N., Ryan B.K, & Kerrigan S.W. (2021). Targeting Internalized Staphylococcus aureus Using Vancomycin-Loaded Nanoparticles to Treat Recurrent Bloodstream Infections. Antibiotics, 10(5), 581.

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Endothelial Cell Culture for Vascular Research

Cited 1 time

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Primary human umbilical vein endothelial cells (HUVEC) were purchased from Cell Applications (200K-05), primary human saphenous vein endothelial cells (HSaVEC) and human aortic endothelial cells (HAoEC) from PromoCell (C-12231 and C-12271, respectively) and human coronary arterial endothelial cells (HCoEC) from Lonza (CC-2585). All cells were cultured in Microvascular Endothelial Cell Growth Medium-2 (complete medium, EGM-2MV, Lonza) and used for no more than 5 passages (16 (link)).

Maier-Begandt D., Comstra H.S., Molina S.A., Krüger N., Ruddiman C.A., Chen Y.L., Chen X., Biwer L.A., Johnstone S.R., Lohman A., Good M.E., DeLalio L.J., Hong K., Bacon H., Yan Z., Sonkusare S.K., Koval M, & Isakson B.E. (2021). A venous-specific purinergic signaling cascade initiated by Pannexin 1 regulates TNFα-induced increases in endothelial permeability. Science signaling, 14(672), eaba2940.

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Cultivation of Human Endothelial Cell Lines

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Human umbilical vein endothelial cells (HUVEC) were purchased from TCS Cellworks or PromoCell and cultured in tissue culture flasks pre-coated with 0.1% gelatin in endothelial cell growth medium (ECGM, PromoCell) supplemented with ECGM-supplement mix and 1% penicillin/streptomycin/amphotericin B (Sigma-Aldrich). HUVEC were used at passages 6–8.
Human cardiac microvascular endothelial cells (HCMEC) were purchased from PromoCell and cultured as described above. HCMEC were used at passages 4–6.
Human Aortic Endothelial Cells (HAoEC) were purchased from Promocell and cultured as described above. HAoEC were used at passages 6–8.

Ahmed S., Kurusamy S., David E.L., Khan K., Kalyanakrishnan K., Ian-Gobo M., Kola T.M., Wilkinson R.N., Kannappan V., Wang W., Gómez M.J., Redondo J.M., Cotton J, & Armesilla A.L. (2022). Aberrant expression of miR-133a in endothelial cells inhibits angiogenesis by reducing pro-angiogenic but increasing anti-angiogenic gene expression. Scientific Reports, 12, 14730.

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Immune Activation of Aortic Endothelial Cells

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Primary human aortic endothelial cells (HAoEC, PromoCell, Germany) were cultured according to manufacturer’s recommendation using the corresponding media as well as the split kit (all, PromoCell, Germany). Cells were used up to passage 8. HAoECs were stimulated for 2 hours with 50 ng/ml IFN-γ, 10 ng/ml TNF-α, or 50 ng/ml IFN-γ + 10 ng/ml TNF-α. Subsequent to the 2-hour incubation, the HAoECs were harvested and RNA was extracted using a Directo-zol RNA MiniPrep (Zymo Research). The extracted RNA was converted to cDNA utilizing the Qiagen RT² Strand Kit and gene expression was analyzed on custom RT-PCR plates for endothelial cell activation markers (Qiagen). Gene expression was reported as fold-change relative to untreated HAoECs.

Mehta N.N., Teague H.L., Swindell W.R., Baumer Y., Ward N.L., Xing X., Baugous B., Johnston A., Joshi A.A., Silverman J., Barnes D.H., Wolterink L., Nair R.P., Stuart P.E., Playford M., Voorhees J.J., Sarkar M.K., Elder J.T., Gallagher K., Ganesh S.K, & Gudjonsson J.E. (2017). IFN-γ and TNF-α synergism may provide a link between psoriasis and inflammatory atherogenesis. Scientific Reports, 7, 13831.

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