Technical note: immunomagnetic procedure for positive selection of macrophages in ovine milk.
A simple immunomagnetic procedure was developed to select macrophages from ovine milk by using a non-specific magnetic positive separation technique. Samples of ewe bulk milk were collected during early, mid, and late lactation; milk samples were centrifuged at 2,000 x g for 30 min at 4 degrees C; the fatty fraction and supernatant were removed, and each pellet was dissolved in 500 microL of pH 7.4 phosphate buffered saline + 0.02% NaN(3). Cells were targeted for selection by using mouse-IgG anti-ovine macrophages. Several trials, testing 2 different fluorochrome-conjugated antibodies [i.e., mouse anti-human CD14:R-Phycoerythrin (RPE) (MCA1568PE, Serotec) and F(ab')2 rabbit anti-mouse IgG:RPE (STAR12A, Serotec)] and 3 different labeling procedures, were performed to evaluate the purity of samples by flow cytometry. A morphological test was carried out by direct microscopic count in enriched fraction smears stained with May-Grünwald-Giemsa stain to confirm the presence of macrophages. The method described in the present technical note can be considered an innovative application to obtain a single-cell population of high purity selected from all the somatic cells in milk.
Caroprese
M
M
Dipartimento PRIME and Istituto per la Ricerca e le Applicazioni Biotecnologiche per la Sicurezza e la Valorizzazione dei Prodotti Tipici e di Qualità (BIOAGROMED), Università di Foggia, Via Napoli 25, 71100 Foggia, Italy. m.caroprese@unifg.it
Marzano
A
A
Schena
L
L
Sevi
A
A
D000818:Animals; D002452:Cell Count; D005260:Female; D005434:Flow Cytometry; D007074:Immunoglobulin G / Q000276:immunology; D018189:Immunomagnetic Separation / Q000379:methods / Q000662:veterinary*; D007774:Lactation; D008264:Macrophages / Q000166:cytology*; D051379:Mice / Q000276:immunology; D008892:Milk / Q000166:cytology*; D012756:Sheep; D013997:Time Factors
D007074:Immunoglobulin G