內皮 Transwell 通透性檢測試劑盒| Endothelial Transwell Permeability Assay Kit貨號CB6929
內皮 Transwell 通透性檢測試劑盒-Endothelial Transwell Permeability Assay Kit貨號CB6929
Endothelial Transwell Permeability Assay Kit (24 SPL PC inserts, 100 assays) Catalog Number: CB6929
Application
定位於血管壁關鍵界面的微血管內皮細胞單層(microvascular endothelial cell monolayer )具有調節組織液平衡( tissue fluid balanc)和提供機體生存所需的必需營養素(essential nutrients)的重要功能。內皮細胞(endothelial cells )能夠動態調節其用於運輸血漿蛋白(transport of plasma proteins)、溶質(solutes)和液體的細胞旁路和跨細胞通路。內皮屏障( endothelial barrier)是一個調節良好的結構，它在正常生理條件下對液體和分子保持最小和選擇性的滲透性。血管滲漏是多種疾病的重要特徵，例如感染性休克(septic shock)、炎症(inflammation)、血腦屏障 (blood-brain barrier,BBB) 相關神經系統疾病的破壞、病毒性出血熱(viral hemorrhagic fever)、癌症和缺血再灌注損傷。迫切需要開發一種高度靈敏和可靠的檢測方法來測量內皮通透性(endothelial permeability)。 Cell Biologics開發了一種簡單、可重複、靈敏、定量的 transwell 通透性測定法來測量內皮屏障功能(endothelial barrier function )，以便在體外對多種類型的內皮通透性( endothelial permeability )進行廣泛分析。該測定將在內皮處理後一小時內通過基於光譜儀的吸光度讀數器（ELISA 板讀數器）在 450 nm 處進行。該檢測試劑盒適用於來自多個物種的初代內皮細胞和永生細胞株。
Storage Condition
Storage Temperature: 4 °C. Kit Components (for 100 assays in 96 well plates): A: SPL PC membrane, 6.5 mm, 0.4 µm pore, white, sterile 24 inserts in a 24-well cell culture plate. B: A 96-well ELISA plate (clear polystyrene wells, flat bottom). C: Streptavidin-HRP solution (150 µl). D: TMB substrate solution (5 ml). E: Stop solution (5 ml)
Procedure All samples and standards should be run in triplicate.
1. Add 1 ml endothelial growth medium in the experiment wells of 24-well plate.
2. About 1~5 x 105 endothelial cells with or without transfection were seeded on the 6.5 mm transwell insert membrane (0.4 µl) in 200~400 µl endothelial growth medium.
3. Transfer the inserts to the wells containing 1 ml of growth medium using a sterilized forceps and cover the plate.
4. Incubate the plate in a cell culture incubator for 2 to 4 days until cells grow by 100% confluence. To check the cell confluence, move the inserts to the empty wells with a sterilized forceps, and wait for 5 minutes to check whether the medium from the upper inserts leaks into the wells of 24-well plate. If there is no leak, then proceed to the next steps. Otherwise, the cells need to grow for days till the cells grow a confluent monolayer.
5. Endothelial cells were starved in a serum-free or 0.1% serum medium for a proper time according to individual experiment purpose.
6. Add the desired stimulus (TNF-, IL-1β, thrombin, LPS, etc.) and 5 µl of streptavidin-HRP to the 300 µl fresh serum-free medium in the top insert chambers.
7. Add 1 ml serum free medium in the wells of a new 24-well cell culture plate and transfer the inserts in to incubate in the 37 oC incubator for a proper treatment time. This assay works best when the cells are treated between 30 min to 24 hours.
8. Transfer 20 µl of media from the lower chamber to the wells of 96-well ELISA assay plate. Each sample would be at least aliquoted in triplicate. No treatment or HRP-free medium were considered as negative control.
9. Add 50 µl TMB substrate into each well of 96-well plate, incubate in a shaker for a couple of minutes at room temperature until the blue color occurred.
10. Add 50 µl stop solution into the wells of 96-well plate, and the blue color will become yellow. Measure the spectrum absorption at 450 nm using an ELISA plate reader. Data expressed as A450 absorption readings are considered a relative permeability.
Authorized Uses of Cell Biologics Products
Endothelial Cell Growth Supplement from Cell Biologics are distributed for internal in vitro research purposes only. Our products are not authorized for human use, for in vitro diagnostic procedures, or for therapeutic procedures. Transfer or resale of any Cell Biologics’ cells or products from the purchaser to other markets, organizations or individuals is prohibited by Cell Biologics, without the company’s written consent. Cell Biologics’ Terms and Conditions must be accepted before submitting an order.
Cell Biologics 是初代培養細胞( primary cultured cells)和細胞培養產品的主要製造商(cell culture products)。提供範圍廣泛的高質量人類和動物初代細胞，包括內皮細胞(endothelial)、上皮細胞( epithelial)、腫瘤細胞( tumor )和幹細胞(stem cells)，以及優化的細胞培養基(optimized cell culture media)和其他相關產品。小鼠初代內皮細胞最初由 Cell Biologics 提供，可根據特定科學需求定制。您可以信賴我們高質量的定制細胞分離服務！該公司還在開發針對炎症性疾病的治療平台和用於治療活性生物製劑的新型遞送系統。
