Flow cytometric assessment of estrogen receptor beta expression in bovine blood neutrophils.
The optimisation of a flow cytometric protocol for the determination of the estrogen receptor beta (ERbeta) expression in bovine blood neutrophils is described. The following final incubation conditions were obtained: fixation with 0.25% formaldehyde and 70% methanol, both for 1 h; permeabilisation with 0.05% Triton X-100, overnight labelling at 4 degrees C with the primary antibody diluted at 10 microg/ml and subsequent labelling for 30 min on ice with the fluorescein isothiocyanate-conjugated secondary antibody at 8 microg/ml. Of the three anti-human or anti-rat ERbeta primary antibodies evaluated, only PA1-311 was found to cross-react with bovine cells. Immunoblot analysis supports the obtained results. The flow cytometric technique allows reproducible quantitative determination of the ERbeta protein in neutrophils and may be a valuable tool for future expression studies in these cells of the innate immune system.
Lamote
Inge
I
Laboratory of Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Demeyere
Kristel
K
Notebaert
Sofie
S
Burvenich
Christian
C
Meyer
Evelyne
E
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D002417:Cattle; D003429:Cross Reactions; D047629:Estrogen Receptor beta / Q000096:biosynthesis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D009504:Neutrophils / Q000378:metabolism*; D051381:Rats
D000906:Antibodies; D047629:Estrogen Receptor beta