A novel, rapid and sensitive heterotypic cell adhesion assay for CD2-CD58 interaction, and its application for testing inhibitory peptides.
The immunoglobulin CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its receptor CD58 on antigen-presenting cells (APCs). Modulation or inhibition of this interaction has been shown to be therapeutically useful. E-rosetting assay is usually applied in the study of the modulation of CD2-CD58 interaction. In this study, we demonstrated a novel, rapid and sensitive heterotypic cell adhesion assay for CD2-CD58 interaction. The CD2 expression on the surface of Jurkat cells and the CD58 expression on the Caco-2 cells were confirmed by flow cytometry and ELISA studies, respectively. Then Jurkat cells were fluorescent-labeled with 2 microM of BCECF-AM for 45 min at 37 degrees C before adding to confluent Caco-2 monolayers cultured in 96-well culture dishes. After 30 min, non-adherent Jurkat cells were removed by washing with PBS, while the monolayer-associated Jurkat cells were lysed with 0.5 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantitated using a microplate fluorescence analyzer with BCECF's excitation maximum of 485 nm and emission maximum of 535 nm. This method was successfully applied for testing inhibitory peptides to CD2-CD58 interaction.
Liu
Jining
J
Department of Pharmacy, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore.
Chow
Vincent T K
VT
Jois
Seetharama D S
SD
D001681:Biological Assay / Q000379:methods*; D018801:CD2 Antigens / Q000276:immunology* / Q000378:metabolism*; D018968:CD58 Antigens / Q000276:immunology* / Q000378:metabolism*; D018938:Caco-2 Cells; D002448:Cell Adhesion / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D019169:Jurkat Cells; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D010835:Phytohemagglutinins / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D012680:Sensitivity and Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D018801:CD2 Antigens; D018968:CD58 Antigens; D010455:Peptides; D010835:Phytohemagglutinins; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate