The use of fluorescent probes as markers for sheep lymphocyte subpopulations

Sheep blood lymphocytes were labelled with fluorescent probes and examined under the fluorescence microscope and by the fluorescence-activated cell sorter. A novel probe using fluorescamine, coupled to hexylamine, detected 22.9% of cells, apparently of the B-cell series, counted by fluorescence microscopy. Substitution of fluorescein isothiocyanate (FITC) for the fluorescamine did not label the same subpopulation of cells although the lymphocytes could then be examined in the cell sorter. A larger number of cells (38.8%) formed the brighter cluster but did not behave as B cell when separated on nylon-wool columns. Improvement in discrimination of the cell populations was obtained with FITC-hexadecylamine (C16). This probe detected 38% of cells in the smallest cluster, 44% of cells in the intermediate cluster and 19% of cells in the brightest cluster. The proportion of cells in each cluster appeared to parallel closely the "null", erythrocyte (E) rosette-forming T cells and the B cells detected by conventional markers for blood lymphocytes. Other fluorescent probes, formed from FITC and other amines and amino acids, labelled lymphocyte membranes. Probes with a terminal charge labelled the small cluster particularly well, whereas those that were terminally non-polar labelled the larger cells brigthly, but not to the same intensity as the charged probes in the small cells.