A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes

• Published in: Journal of immunological methods Vol. 178; no. 1; pp. 41 - 51
• Main Authors: Braut-Boucher, Françoise, Pichon, Jacqueline, Rat, Patrice, Adolphe, Monique, Aubery, Michèle, Font, Jacqueline
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 1995; Elsevier
• Subjects: Biological and medical sciences; Calcein AM; Cell Adhesion; Cell interactions, adhesion; Cell-cell adhesion; Cells, Cultured; Cytological Techniques; Diverse techniques; Fibroblasts - physiology; Fluoresceins; Fluorescent Dyes; Fluorimetric microtitration assay; Fluorometry - methods; Fundamental and applied biological sciences. Psychology; Humans; Isotope Labeling; Keratinocyte; Keratinocytes - physiology; Lymphocyte; Lymphocytes - physiology; Molecular and cellular biology; PBS; DMSO; EGF; PMA; Cell-cell adhesion; BCECF; EDTA; Fluorimetric microtitration assay; FU; mAb; BSA; Calcein AM; FCS; Keratinocyte; CLF; Lymphocyte; ELISA; Assay; Human; Sensitivity; Fluorescent tracer; Adhesion; Fluorescence spectrometry
• ISSN: 0022-1759; 1872-7905
• 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 μM) during a short incubation period (30 min); the adhesion of 2 × 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.