Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J
INSERM U180, UFR Biomédicale des Saints-Pères, Université René-Descartes, Paris, France.
J Immunol Methods. 1995 Jan 13;178(1):41-51. doi: 10.1016/0022-1759(94)00239-s.
A simple and sensitive cell-cell adhesion microplate assay was established using the cytoplasmic fluorescent dye, calcein AM. The procedure involves three steps: the labeling of lymphocytes with an adequate concentration of calcein AM (20 microM) during a short incubation period (30 min); the adhesion of 2 x 10(5) labeled lymphocytes per well to confluent keratinocyte or fibroblast monolayers grown in microtiter plates for 90 min; and, finally, measurement of the fluorescent signal utilizing a new system of cold-light microfluorimetry (Rat, 1993). During the adhesion assay, the release of calcein from labeled lymphocytes is low and the method permits the detection of as few as 1000 adherent cells. This non-radioactive procedure takes less than 4 h to perform and has proven to be as accurate and reliable as the common method using radioactive isotopes. In addition to its simplicity, the use of a fluorescent molecular probe in conjunction with cold-light microfluorimetry (CLF) offers many advantages of safety and economy, and can readily be adapted to the different cell types that participate in cell-cell adhesion.
利用细胞质荧光染料钙黄绿素乙酰甲酯（calcein AM）建立了一种简单且灵敏的细胞间黏附微孔板检测方法。该方法包括三个步骤：在短时间孵育期（30分钟）内用适当浓度（20微摩尔）的钙黄绿素乙酰甲酯标记淋巴细胞；每孔2×10⁵个标记淋巴细胞与在微孔板中生长的汇合角质形成细胞或成纤维细胞单层黏附90分钟；最后，利用一种新型冷光显微荧光测定系统测量荧光信号（拉特，1993年）。在黏附检测过程中，标记淋巴细胞中钙黄绿素的释放量很低，该方法能够检测到低至1000个黏附细胞。这种非放射性方法执行时间不到4小时，并且已证明与使用放射性同位素的常用方法一样准确可靠。除了操作简单外，荧光分子探针与冷光显微荧光测定法（CLF）结合使用具有许多安全和经济优势，并且可以很容易地应用于参与细胞间黏附的不同细胞类型。
