CytoSelect™ 96-well Leukocyte-endothelium Adhesion Kit
(19 references)-
- 检测方法
- Fluorometric
- 应用范围
- Biochemical Assay (BCA)
- 品牌
- CytoSelect™
- 样品类型
- Cell Samples, Serum
- Analytical Method
- Quantitative
- 产品特性
- CytoSelect™ Leukocyte-endothelium Adhesion Assay provides a robust system for the quantitative determination of leukocyte-endothelium interactions. The kit contains sufficient reagents for the evaluation of 100 assays in a 96-well plate.
- 组件
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- 500X LeukoTracker™ Solution : One 100 μL tube
- Gelatin Solution : One 12 mL bottle of sterile 0.1 % Gelatin in 1X PBS
- 4X Lysis Buffer : One 10 mL bottle 3
- 10X Wash Buffer : One 20 mL bottle
- TNFα : One 100 μL tube of 10 μg/mL TNFα in sterile 1X PBS/0.1%BSA
- 试剂未包括
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- Endothelial cells and cell culture medium
- 96-well or 48-well plate
- Serum free medium, such as DMEM containing 0.5 % BSA, 2 mM CaCl2 and 2 mM MgCl2
- Sterile 1X PBS
- Cell culture incubator (37 °C, 5 % CO2 atmosphere)
- Light microscope
- 96-well plate suitable for a fluorescence plate reader
- Fluorescence plate reader
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Tumor Necrosis Factor alpha (TNF alpha) ELISA Kit
TNF alpha 适用: 大鼠 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Interleukin 1, beta (IL1B) ELISA KitIL1B 适用: 大鼠 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Tumor Necrosis Factor alpha (TNF alpha) ELISA KitTNF alpha 适用: 小鼠 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
High Mobility Group Box 1 (HMGB1) ELISA KitHMGB1 适用: 人 Colorimetric Sandwich ELISA 62.5 pg/mL - 4000 pg/mL Plasma, Serum
Lipopolysaccharides (LPS) ELISA Kit适用: Various Species Colorimetric Competition ELISA 12.35 ng/mL - 1000 ng/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Tumor Necrosis Factor alpha (TNF alpha) ELISA KitTNF alpha 适用: 人 AA 77-233 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Plasma (EDTA), Plasma (citrate), Plasma (heparin), Serum
Interleukin 6 (IL6) ELISA KitIL6 适用: 人 Colorimetric Sandwich ELISA 7.8-500 pg/mL Cell Culture Supernatant, Plasma, Serum, Tissue Homogenate, Urine
Vascular Cell Adhesion Molecule 1 (VCAM1) ELISA KitVCAM1 适用: 人 Colorimetric Sandwich ELISA 1.563-100 ng/mL Plasma, Serum, Tissue Homogenate
Matrix Metalloproteinase 2 (MMP2) ELISA KitMMP2 适用: 人 Colorimetric Sandwich ELISA 0.78 ng/mL - 50 ng/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Interleukin 1, beta (IL1B) ELISA KitIL1B 适用: 人 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
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- 应用备注
- Optimal working dilution should be determined by the investigator.
- 说明
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- Quantify interactions between leukocytes and the endothelium
- Fully quantitative with no manual cell counting
- Highly sensitive results on a fluorescence plate reader
- 试剂准备
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1X Wash Buffer: Prepare a 1X Wash Buffer by diluting the provided 10X stock 1:10 in deionized water. Store the diluted solution at room temperature. 1X Lysis Buffer: Prepare a 1X Lysis Buffer by diluting the provided 4X stock 1:4 in deionized water. Store the diluted solution at room temperature. Gelatin Coating 1. Under sterile conditions, add 200 μL of the Gelatin Solution to each well of a 48-well tissue culture treated plate, or 100 μL of the Gelatin Solution to each well of a 96-well tissue culture treated plate. 2. Incubate for 60 min at 37 °C in a cell culture incubator. 3. Wash twice with sterile 1X PBS. Aspirate the final wash before use.
- 实验流程
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- Add 50,000-100,000 endothelial cells/well to the Gelatin-coated 48-well or 96-well plate.
- Culture cells for 48-72 until the endothelial cells form a monolayer.
- Treat endothelial cell monolayer or leukocyte with desired activator or inhibitor for 6-12 hrs. 4 6
- Harvest leukocytes and prepare a cell suspension at 1.0 x 10 cells/mL in serum free media. Add LeukoTracker to a final concentration of 1X (for example, add 2 μL of 500X LeukoTracker™ solution to 1.0 mL of leukocyte cell suspension).
- Incubate for 60 min at 37 °C in a cell culture incubator. Spin down cells at 1000 rpm for 2 minutes, aspirate the medium and wash cell pellet with serum free media. Repeat the wash twice. 6 Resuspend the cell pellet at 0.25 - 1.0 x 10 cells/mL in serum free media.
- Aspirate endothelial culture media and wash once with serum free media. Add 200 μL of the cell suspension to each well already containing the endothelial monolayer.
- Incubate for 30-90 min in a cell culture incubator.
- Carefully discard or aspirate the media from each well (Note: Do not allow wells to dry). Gently wash each well 3 times with 250 μL 1X Wash Buffer.
- (Optional) Count the adherent leukocytes under an inverted fluorescence microscope, average at least three separate fields per well.
- Aspirate the final wash and add 150 μL of 1X Lysis Buffer to each well containing cells. Incubate 5 minutes at room temperature with shaking.
- Transfer 100 μL of the mixture to a 96-well plate suitable for fluorescence measurement. Read fluorescence with a fluorescence plate reader at 480 nm/520 nm.
- 限制
- 仅限研究用
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- 储存条件
- 4 °C/-20 °C
- 储存方法
- LeukoTracker™ Solution and TNFa should be removed from the kit and stored at -20°C immediately. Store all other components at 4°C.
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: "Poliovirus Receptor-Related 2: A Cholesterol-Responsive Gene Affecting Atherosclerosis Development by Modulating Leukocyte Migration." in: Arteriosclerosis, thrombosis, and vascular biology, Vol. 37, Issue 3, pp. 534-542, (2017) (PubMed).
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- 背景
- Leukocyte extravasation into perivascular tissue plays a key role in inflammatory diseases. This recruitment requires leukocyte interaction with vascular endothelium and consists of multiple, consecutive processes including the capture of circulating leukocytes, subsequent leukocyte rolling, arrest, firm adhesion and transmigration (Figure 1). This multistep paradigm is realized by sequential activation-dependent interactions between endothelial cell adhesion molecules and their specific ligands on leukocytes. The first step of transient adhesion and rolling is known to be mediated by an interaction of leukocyte or endothelial cell selectins and their oligosaccharide-bearing ligands. Arrest and firm adhesion of leukocytes to endothelium is dependent on the activation of β2 integrins like Mac-1 or LFA-1 on the leukocyte cell surface, followed by interaction with endothelial cell proteins belonging to the Ig superfamily such as ICAM-1.
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