Weitz-Schmidt Gabriele, Chreng Stéphanie
Novartis Institutes for BioMedical Research, Basel, Switzerland.
Methods Mol Biol. 2012;757:15-30. doi: 10.1007/978-1-61779-166-6_2.
Standard adhesion assays measure cell binding either to immobilized ligands or to cell monolayers in flat-well microtiter plates under static conditions. Typically, these test systems require several washing steps to separate adherent from nonadherent cells. Here, we describe an adhesion assay which avoids these washing steps by employing V-bottom 96-well plates. In this assay, fluorescently labeled leukocytes are allowed to adhere to V-well plates coated with soluble ligand for a fixed time. Thereafter, centrifugal force is applied to separate adherent cells from nonadherent cells. Nonadherent cells accumulate in the nadir of the V-shaped wells and are quantified using a fluorometer with a narrow aperture. This simple and reproducible method has been validated with different classes of adhesion molecule families (selectins and integrins) and is adaptable to several other adhesive interactions. The assay format is suitable for screening applications and may also be used for diagnostic testing. The receptor/ligand interaction chosen as an example to describe the assay methodology is the interaction between the integrin lymphocyte function-associated molecule-1 (LFA-1, α(L)β(2)) and intercellular adhesion molecule-1 (ICAM-1).
标准黏附试验在静态条件下测量细胞与固定化配体或平底微孔板中的细胞单层的结合。通常，这些测试系统需要几个洗涤步骤来分离贴壁细胞和非贴壁细胞。在此，我们描述一种黏附试验，该试验通过使用V型底96孔板避免了这些洗涤步骤。在该试验中，使荧光标记的白细胞在固定时间内黏附于包被有可溶性配体的V型孔板。此后，施加离心力以分离贴壁细胞和非贴壁细胞。非贴壁细胞聚集在V型孔的最低点，并使用具有窄孔径的荧光计进行定量。这种简单且可重复的方法已通过不同类别的黏附分子家族（选择素和整合素）得到验证，并且适用于其他几种黏附相互作用。该试验形式适用于筛选应用，也可用于诊断测试。作为描述试验方法的示例选择的受体/配体相互作用是整合素淋巴细胞功能相关分子-1（LFA-1，α(L)β(2)）与细胞间黏附分子-1（ICAM-1）之间的相互作用。
