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Published on: June 23, 2013
Technical note: immunomagnetic procedure for positive selection of macrophages in ovine milk
M Caroprese1, A Marzano, L Schena
1Dipartimento 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
Abstract:
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.
Insights
This study presents a new immunomagnetic method for isolating pure macrophage populations from ewe milk. This technique enhances cell separation for research and diagnostic applications in ovine mastitis studies.
Area of Science:
- Veterinary Immunology
- Cell Biology
- Mammalian Physiology
Background:
- Macrophages are crucial immune cells in milk, playing a role in mammary gland health and disease.
- Isolation of pure macrophage populations from milk is challenging due to the presence of other somatic cells.
- Understanding macrophage function in ovine milk requires reliable isolation techniques.
Purpose of the Study:
- To develop and validate a simple immunomagnetic procedure for selecting macrophages from ovine milk.
- To assess the purity and efficacy of the developed isolation method.
Main Methods:
- Ovine milk samples were collected during different lactation stages.
- Immunomagnetic separation using mouse IgG anti-ovine macrophages was employed.
- Flow cytometry and morphological analysis (May-Grünwald-Giemsa stain) were used to evaluate cell purity.
Main Results:
- A high purity of macrophages was achieved using the immunomagnetic technique.
- The method successfully isolated macrophages from the complex somatic cell population in milk.
- Optimized antibody labeling and separation procedures confirmed macrophage presence.
Conclusions:
- The developed immunomagnetic procedure offers an innovative and effective way to obtain pure ovine milk macrophages.
- This method provides a valuable tool for studying macrophage biology in the context of ovine mammary health.
- The technique has potential applications in research and diagnostics related to ovine mastitis.
