Anti‐kinetochore staining for single laser, bivariate flow sorting of Indian muntjac chromosomes

Flow cytometric chromosome sorting typically relies upon dual‐laser, bivariate analysis after staining with two different base pair‐specific dyes for resolution of chromosomes with similar DNA content. The availability of FITC‐conjugated antibodies offers the possibility of singlelaser bivariate analysis when combined with propidium iodide (PI) DNA staining, but requires exploitable antigenic differences between chromosomes of interest. A technique was developed for indirect immunofluorescent anti‐kinetochore staining of Indian muntjac chromosomes in suspension. Primary antibody binding within permeabilized whole cells minimized centrifugation‐induced loss of chromosomal integrity. Subsequent FITC‐conjugated second antibody binding was not affected by concurrent PI‐counterstaining. Anti‐kinetochore staining facilitated resolution of chromosomes No. 2 and X, which formed a doublet peak upon univariate DNA content analysis, as well as recognition of the Y2 peak which was indistinguishable from debris by univariate analysis. The technique allowed greater than 90% purification of each Indian muntjac chromosome.