A simple quantitative in vitro assay for thymocyte adhesion to thymic epithelial cells using a fluorescein diacetate

Recent data verified the role of thymocyte adhesion to thymic epithelial cells (TEC) in T cell development. In order to measure this cellular event as one of routine examinations in immunological studies, a simple quantitative assay in in vitro setting is required. Labeling thymocytes with fluorescein diacetate (FDA) was used to measure quickly and with certainty the adhesion ability of BALB/c thymocytes to TEC cells, a thymic epithelial cell line derived from BALB/c mouse thymic stroma. Thymocytes stained with FDA at the concentration of 2.5 micrograms/ml gave a strong fluorescent intensity (FI) easily detectable in a spectrophotometer and showed the same phenotypical and functional features as unstained cells. As it was confirmed that the cell number correlated well with FI, the number of cells adhering to TEC in wells of a 96-well microplate could be estimated. The major advantage of using FDA was that it took only a few seconds to measure FI of one sample. In contrast, to count the number of adherent cells with conventional methods using a light microscope or radioisotopes required much longer time and care to avoid radiological hazards. Moreover, cell viability and FI of FDA-labeled thymocytes changed little for 24 h, and lysates of FDA-positive cells demonstrated the same FI as living cells. These results indicate that many samples could be applied for FI measurement at the same time after in vivo or in vitro experiments.