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 single-laser 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.
Levy
H P
HP
Division of Human Genetics, University of Maryland, Baltimore.
Schultz
R A
RA
Ordóñez
J V
JV
Cohen
M M
MM
D000818:Animals; D002460:Cell Line; D002469:Cell Separation; D002875:Chromosomes / Q000648:ultrastructure*; D004247:DNA / Q000032:analysis; D003670:Deer; D005347:Fibroblasts / Q000648:ultrastructure*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007621:Karyotyping / Q000379:methods*; D011419:Propidium; D012598:Sclerosis / Q000276:immunology; D013194:Staining and Labeling
D011419:Propidium; D004247:DNA; D016650:Fluorescein-5-isothiocyanate