The Mouse T Lymphocyte Activation Antibody Cocktail is a three-color reagent designed to identify activated T lymphocytes by direct immunofluorescent staining with flow cytometric analysis. This cocktail consists of the following antibody mixture: PE-Cy7 rat anti-mouse CD25 (clone PC61), PE hamster anti-mouse CD69 (clone H1.2F3), and APC hamster anti-mouse CD3e (clone 145-2C11). The PC61 antibody reacts with CD25, the low-affinity IL-2 Receptor achain (IL-2Rα, p55) expressed on activated T and B lymphocytes from all mouse strains tested. CD25 is also found on some developing B cells in the bone marrow, early developing T cells in the thymus, peripheral CD4+ regulatory T (Treg) cells, and dendritic cells. The H1.2F3 antibody reacts with CD69 (Very Early Activation antigen). Its expression is rapidly induced upon activation of lymphocytes (T, B, NK, and NK-T cells) neutrophils, and macrophages. CD69 is expressed also on thymocytes that are undergoing positive selection. The 145-2C11 antibody reacts with the 25-kDa echain of the T-cell receptor-associated CD3 complex, which is expressed on thymocytes, mature T lymphocytes, and NK-T cells of all mouse strains tested. The three antibodies have been titrated and pre-diluted, mixed together, and formulated for optimal staining performance. The Mouse T Lymphocyte Activation Isotype Control contains equivalent concentrations of fluorochrome- and isotype-matched negative-control immunoglobulins.
The use of three different fluorochromes for the labeling of the three different antibodies permits the recognition of each of the three antigens on each cell in a sample. The expression of the three antigens identifies activated T lymphocytes. Additional fluorochrome-labeled reagents may be combined with the Mouse T Lymphocyte Activation Antibody Cocktail, and the Mouse T Lymphocyte Activation Isotype Control, to further characterize activated T-cell subpopulations.
The Mouse T Lymphocyte Activation Isotype Control contains equivalent concentrations of fluorochrome- and isotype-matched negative-control immunoglobulin consisting of the following: PE-Cy™7 rat IgG1, λ (clone A110-1), PE Armenian hamster IgG1, λ (clone G235-2356) and APC Armenian hamster IgG1, κ (clone A19-3).