The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells.
A flow cytometric technique was developed to measure the relative concentration of whey protein and beta-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (alpha-lactalbumin + beta-lactoglobulin) or beta-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more beta-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.
Keys
J E
JE
USDA-ARS, Milk Secretion and Mastitis Laboratory, Beltsville, Maryland 20705, USA.
Guidry
A J
AJ
Cifrian
E
E
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002364:Caseins / Q000032:analysis / Q000276:immunology; D002417:Cattle; D002460:Cell Line; D004848:Epithelium / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007768:Lactalbumin / Q000032:analysis / Q000276:immunology; D007782:Lactoglobulins / Q000032:analysis; D008321:Mammary Glands, Animal / Q000737:chemistry*; D008894:Milk Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity
D000906:Antibodies; D002364:Caseins; D005456:Fluorescent Dyes; D007782:Lactoglobulins; D008894:Milk Proteins; D007768:Lactalbumin; D016650:Fluorescein-5-isothiocyanate