Expression of VCAM-1 on the surface of HUVECs was evaluated by incubating 2 × 105 HUVECs in EGM in the absence or presence of 20 ng ml−1 human TNFα (hTNFα Millipore, Billerica, MA, USA) for 24 h. After harvesting and washing with phosphate-buffered saline solution (PBS), the cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature. After blocking with PBS containing 1% bovine serum albumin (BSA) for 1 h at room temperature, the cells were incubated first with mouse anti-VCAM-1 monoclonal antibody (1 μg ml−1; Abcam, Cambridge, UK) for 1 h at 37 °C, and then with Alexa Fluor 488-conjugated anti-mouse antibody (1:1000; Invitrogen) for 1 h at 37 °C. The effects of anti-VCAM-1-D6 IgG on endothelial cell activation were evaluated by incubating 2 × 105 HUVECs in the absence or presence of 20 ng ml−1 hTNFα (Millipore), 20 μg ml−1 control IgG or anti-VCAM-1-D6 IgG for 24 h. After blocking with PBS containing 1% BSA for 1 h at room temperature, the cells were incubated first with rabbit anti-ICAM-1 monoclonal antibody (1:500; Abcam) for 1 h at 37 °C, and then with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:1000; Invitrogen) for 1 h at 37 °C. Samples were analyzed by flow cytometry using a FACSCalibur system (BD Biosciences, San Jose, CA, USA) with the aid of FlowJo software (TreeStar, Ashland, OR, USA).
Htnf α
Manufactured by Merck Group
Sourced in United States
HTNF-α is a laboratory reagent used in scientific research. It is a recombinant human tumor necrosis factor alpha protein. This protein is a key mediator of the inflammatory response and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Bio-Techne (1 mentions)
Hil 4, by R&D Systems (1 mentions)
Elispot assay kit, by R&D Systems (1 mentions)
Hgm csf, by Merck Group (1 mentions)
Eb h209, by Thermo Fisher Scientific (1 mentions)
Recombinant pifn γ, by R&D Systems (1 mentions)
Y 27632, by Merck Group (1 mentions)
Parthenolide, by Merck Group (1 mentions)
Haecs, by Lonza (1 mentions)
Alexa fluor 488 conjugated anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti vcam 1 monoclonal antibody, by Abcam (1 mentions)
Facscalibur system, by BD (1 mentions)
Alexa fluor 647 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Icam 1, by Abcam (1 mentions)
Vcam 1, by Abcam (1 mentions)
5 protocols using htnf α
1
Evaluating VCAM-1 Expression on HUVECs
2
Dendritic Cell Differentiation and Transfection
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About 4x106 THP-1 cells were seeded in T75 flasks and differentiated towards immature dendritic cells (iDC) over a 5-day culture in 20 mL serum-supplemented RPMI 1640 in the presence of 100 ng/mL hIL4 (R&D Systems, 204-IL-020/CF) and 100 ng/mL hGM-CSF (Sigma-Aldrich, #GF304). To allow full maturation towards mature dendritic cells (mDC), iDC were collected via centrifugation, resuspended in serum free RPMI 1640 media supplemented with 200ng/mL hIL4, 100ng/mL hGM-CSF, 20ng/mL hTNFα (Sigma-Aldrich, #GF314), and 200ng/mL ionomycin (Tocris Bioscience, 2092/1), and plated at density 10,000/well in the 96-well plates provided with the ELISPOT assay kit R&D Systems, #EL485, Minneapolis, MN. Cells were kept in culture for 1 day to allow differentiation towards mDC, before beginning the transfection with MessengerMax-formulated mRNA to induce expression of the E6-E7 fusion protein in the vaccine. The day following the transfection, human antigen specific E711-20 T cells (Charles River Laboratories, #ASTC-1099) were added to the 96-well plate at density 20,000 cells/well. ASTC and mDC cells were kept in coculture for an overnight. The plates were processed as per manufacturer (R&D Systems, #EL485)’s instructions and read under the CTL Immunospot Analyzer (ImmunoSpot®). CD209 monoclonal antibody eB-h209 (eBioscience™) was used to characterize DC differentiation via Flow Cytometry.
3
Activation of pCECs and Monocyte Assay
4
Activation of Aortic Endothelial Cells
5
Evaluating K202.B Effects on Endothelial Activation
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The effects of K202.B on endothelial cell activation were evaluated by incubating 2 × 105 HUVECs with or without 20 ng/mL of human tumor necrosis factor-α (hTNFα; Millipore), 20 μg/mL of K202.B, or control IgG for 24 h. The cells were fixed with 4% (v/v) PFA in PBS and incubated with 10 μg/well of intercellular cell adhesion molecule-1 (ICAM-1; Abcam, Cambridge, MA, USA) or vascular cell adhesion molecule-1 (VCAM-1; Abcam) antibody for 1 h at 25 °C. Then, Alexa Fluor 647-conjugated anti-mouse IgG or antirabbit IgG (1:1000; Invitrogen) was incubated for 1 h at 25 °C. All samples were analyzed using flow cytometry with the aid of FlowJo software (TreeStar, Ashland, OR, USA).
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