Vcam 1

Manufactured by Thermo Fisher Scientific

Sourced in United States, Austria

VCAM-1 is a cell adhesion molecule that plays a role in the inflammatory response. It is expressed on the surface of endothelial cells and is involved in the adhesion of leukocytes to the vascular endothelium.

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42 protocols using vcam 1

Adhesion Assay for Integrin-Mediated Cell Attachment

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Fibronectin (10 μg/ml, Corning) or VCAM-1 (10 μg/ml, R&D Systems) coated wells were blocked with 10 mg/ml heat-denatured BSA for 30 minutes. For adhesion to Fibronectin, cells were allowed to attach for 10 minutes at 37°C. Adherent cells were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet solution. The dye was solubilized in 10% acetic acid, and absorbance measured at 570 nm. For adhesion to VCAM-1, cells were incubated with Hoechst 33342 (5 μg/ml; Thermo Fisher Scientific) for 10 minutes before plating on VCAM-1-coated wells for 30 minutes at 37°C. Adherent cells were imaged and analyzed using an ImageXpress high-content analysis system (Molecular Devices). For antibody blocking experiments, cells were incubated with anti-α4 integrin antibody (clone PS/2, 10 μg/ml; Abcam) for 1 hour at room temperature before attachment to VCAM-1.

Sarvi S., Patel H., Li J., Dodd G.L., Creedon H., Muir M., Ward J., Dawson J.C., Lee M., Culley J., Salter D.M., Sims A.H., Byron A, & Brunton V.G. (2018). Kindlin-1 promotes pulmonary breast cancer metastasis. Cancer research, 78(6), 1484-1496.

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Quantifying Vascular Receptor Expression

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To investigate the relative expression of EC receptors in different organs, we intravenously injected 20 μg of antibodies against E-selectin, P-selectin (BD PharMingen), ICAM1, ICAM2, PECAM1, CD36 (BioLegend) and VCAM1 (Invitrogen) conjugated to FITC (VCAM1) or A647 (all other antibodies), into uninfected or day 6 infected mice, as previously described in the context of parasitology for CD31 (Hopp et al., 2015 ). We measured MFIs of at least 100 different vessels per organ in 3 separate mice using an LSM 710 Zeiss microscope, a 40x objective (1.3 NA).

De Niz M., Brás D., Ouarné M., Pedro M., Nascimento A.M., Henao Misikova L., Franco C.A, & Figueiredo L.M. (2021). Organotypic endothelial adhesion molecules are key for Trypanosoma brucei tropism and virulence. Cell Reports, 36(12), 109741.

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Quantitative Real-time PCR of Aortic Genes

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Quantitative Real‐time PCR was performed using aortic tissue and cell lysate as previously described.23, 26 The primers for glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh), Icam‐1, pre‐miR‐34a, miR‐34a, monocyte chemoattractant protein 1 (Mcp‐1), P53, selectin E (Sele), Sirt1, U6 and Vcam‐1 were obtained from Life Technologies (Shanghai, China).

Wu J., Liang W., Tian Y., Ma F., Huang W., Jia Y., Jiang Z, & Wu H. (2019). Inhibition of P53/miR‐34a improves diabetic endothelial dysfunction via activation of SIRT1. Journal of Cellular and Molecular Medicine, 23(5), 3538-3548.

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Quantitative Real-Time PCR of Aortic Tissue

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Aortic tissue or cell lysates were used for quantitative real-time PCR, the procedure of which was described previously (Wu et al. 2014) (link). The primers for glyceraldehyde 3-phosphate dehydrogenase (Gapdh), Icam-1, monocyte chemoattractant protein 1 (Mcp-1), P53 and Vcam-1 were purchased from Life Technologies.

Wu H., Wu J., Zhou S., Huang W., Li Y., Zhang H., Wang J, & Jia Y. (2018). SRT2104 attenuates diabetes-induced aortic endothelial dysfunction via inhibition of P53. The Journal of endocrinology, 237(1).

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Quantitative RT-PCR for Keratinocyte Genes

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Cutaneous tissues or cell lysates of keratinocytes were used for quantitative RT-PCR, as described previously (Wu et al. 2014b) . The primers for Gapdh, Ho1, intercellular adhesion molecule-1 (Icam-1), Nqo1 and Vcam-1 were provided by Life Technologies.

Li Y., Ma F., Li H., Song Y., Zhang H., Jiang Z, & Wu H. (2018). Dimethyl fumarate accelerates wound healing under diabetic condition. Journal of molecular endocrinology, 61(4).

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Quantitative Real-Time PCR Analysis

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Quantitative real-time PCR was performed as previously described (Wang et al. 2009 (link), Wu et al. 2014) (link). Primers for collagen 4 (Col4), Fn, Gapdh, Nqo1, intercellular adhesion molecule 1 (Icam-1), Ho1, iNos, Keap1, Nrf2 and Vcam-1 were all purchased from Life Technologies.

Zhang H., Liu X., Zhou S., Jia Y., Li Y., Song Y., Wang J, & Wu H. (2018). SP600125 suppresses Keap1 expression and results in NRF2-mediated prevention of diabetic nephropathy. Journal of molecular endocrinology, 60(2).

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MDSC Migration Assay in Endometriosis

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HTS Transwell®−24 well plates (3μm, Corning Inc., Corning, NY, USA) were used in the assay of MDSC migration in vitro. Control peritoneal fluid and control serum samples were obtained from naive mice, whereas endometriosis peritoneal fluid and endometriosis serum samples were obtained from mice at 12h after transplantation of endometrial implants. MDSCs were isolated from the peritoneal cells in mice at 12h after transplantation of endometrial implants. Control peritoneal fluid, endometriosis peritoneal fluid, control serum and endometriosis serum as well as 10 ng/mL of recombinant murine VCAM-1, MIP-1γ, CXCL-1, CXCL-2, CXCL-5, G-CSF, MCP-1, C10 and C5/C5a (PeproTech Inc., Rocky Hill, NJ, USA) were added in the bottom chamber of transwell plates. MDSCs (2×10⁵) were seeded each well in the upper chamber of transwell plates. CXCR2 inhibitors, SB 265610 and SB225002 (R&D Systems Inc., Minneapolis, MN, USA) were also added in the upper chamber for inhibition assay. After incubation for 4 h, migrated cells were harvested and counted under microscope using trypan blue staining. The percentages of migrated MDSCs were calculated.

Zhang T., Zhou J., Man G.C., Leung K.T., Liang B., Xiao B., Ma X., Huang S., Huang H., Hegde V.L., Zhong Y., Li Y., Kong G.W., Yiu A.K., Kwong J., Ng P.C., Nagarkatti P.S., Nagarkatti M, & Wang C.C. (2018). MDSCs drive the process of endometriosis by enhancing angiogenesis and provide a new potential approach for treatment. European journal of immunology, 48(6), 1059-1073.

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Immunofluorescence Analysis of Cell Markers

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The mouse antibody anti-H3K9me2/3 (#5327), and the rabbit antibody -phospho-ERM (#3726) were from Cell Signaling (Danvers, MA, USA). The mouse antibody anti-β-tubulin (#T5201) was from Sigma-Aldrich. The antibody anti-α4 (HP2/1) was a gift from Prof. Sánchez-Madrid (Hospital de la Princesa, Universidad Autónoma de Madrid). The anti-ZO-1 antibody was from SantaCruz (Santa Cruz, CA, USA) (#sc-33725). Tetramethylrhodamine (TRITC)-Phalloidin, Alexa 647-Phalloidin, CellTrace™ CFSE, secondary antibodies Alexa-488, -594, -647 for immunofluorescence analysis and DAPI were obtained from Thermo Scientific (Waltham, MA, USA). VCAM1 was obtained from Peprotech. BIX01294 was from Abcam (Cambridge, UK).

Madrazo E., Ruano D., Abad L., Alonso-Gómez E., Sánchez-Valdepeñas C., González-Murillo Á., Ramírez M, & Redondo-Muñoz J. (2018). G9a Correlates with VLA-4 Integrin and Influences the Migration of Childhood Acute Lymphoblastic Leukemia Cells. Cancers, 10(9), 325.

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Intraperitoneal Injection of Immune Modulators

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Peritoneal fluid and serum samples from naive mice and mice with endometriosis, recombinant murine VCAM-1, MIP-1 gamma, CXCL-1, CXCL-2, CXCL-5, G-CSF, MCP-1, C10 and C5/C5a (PeproTech Inc., Rocky Hill, NJ, USA) were intraperitoneally injected into naive mice. Peritoneal cells were harvested at 24 hours for flow cytometry analysis. The percentage and number of MDSCs in the peritoneal cavities of mice were calculated and compared among different treatment groups.

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RT-qPCR Analysis of Inflammatory Genes

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RNA was isolated from treated HUVECs according to the manufacturer’s specifications (Qiagen) and reverse transcribed (Applied Biosystems). Respective cDNA was amplified in a CFX Realtime System (Biorad) at a volume of 20 μL containing dilutions of 1:20 Taqman probe (Applied Biosystems), 1:2 Taqman Gene Expression Master Mix (Applied Biosystems), and 1:10 cDNA in ddH₂O. RT-PCR gene probes were purchased from Applied Biosystems and included: CCL5 (#Hs00174575_m1), CCL20 (#Hs01011368_m1), IL6 (#Hs00985639_m1), SELE (#Hs00950401_m1), VCAM-1 (#Hs01003372_m1), and GAPDH (Hs02758991_g1). For amplification, samples were heated to 50 °C for 2 min for once cycle, 95 °C for 10 min for one cycle, and then 40 cycles where samples were heated to 95 °C for 15 s proceeded by 60 °C for 1 min. Cycle times were subsequently normalized to the average GAPDH cycle time for all samples such that a relative decrease in cycle number by x indicates gene induction by 2^x.

Fang C., Manes T.D., Liu L., Liu K., Qin L., Li G., Tobiasova Z., Kirkiles-Smith N.C., Patel M., Merola J., Fu W., Liu R., Xie C., Tietjen G.T., Nigrovic P.A., Tellides G., Pober J.S, & Jane-wit D. (2019). ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes. Nature Communications, 10, 2247.

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