Colorimetric assay for the measurement of thymocyte/thymic stromal cell adhesion.
1. We describe a simple and accurate colorimetric adhesion assay (CAA) and illustrate the assay by measuring the adhesion of mouse thymocytes to mouse 2BH4 cells. 2. The assay is based on the crystal violet staining of thymocytes adhered to a subconfluent layer of 2BH4 cells (plated at 2 x 10(4) cells/well for 24 h). The optimal incubation time was shown to be 1 h and washing in PBS of non-bound and non-specifically bound thymocytes is the critical step for the precision and accuracy of the assay. 3. Saturation curves were obtained for thymocytes adhered to plated 2BH4 cells. The blank (only 2BH4 cells) was near 0.200 +/- 0.010 (mean +/- SD) and was quite reproducible. As expected, the extent of adhesion was also dependent on the number of plated 2BH4 cells. Standard curves need to be run with each assay for quantitative measurements. The intra-assay and interassay coefficients of variation were 5% and 20%, respectively. 4. The specificity of the reaction was demonstrated by the reduction of adherence by trypsin pretreatment of thymocytes, and the dose-dependent inhibition of adherence by rabbit anti-mouse thymocyte antisera but not rabbit anti-mouse immunoglobulin antisera. 5. The proposed method is simple and requires less effort than the counting of adhered cells with the light microscope and does not require the use of radioactive material as when labelling with Na2(51)CrO4 is utilized.
Amarante-Mendes
J G
JG
Departamento de Imunologia, Universidade de São Paulo, Brasil.
Starobinas
N
N
Macedo
M S
MS
D000704:Analysis of Variance; D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D003124:Colorimetry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D013194:Staining and Labeling; D017154:Stromal Cells; D013950:Thymus Gland / Q000166:cytology*