Evaluation of elutriation and magnetic microbead purification of canine monocytes

• Published in: Veterinary immunology and immunopathology Vol. 101; no. 3; pp. 171 - 178
• Main Authors: de Carvalho, Camila Miranda, Bonnefont-Rebeix, Catherine, Picandet, Stephanie, Bernaud, Janine, Phothirath, Phoukham, Chabanne, Luc, Marchal, Thierry, Magnol, Jean-Pierre, Rigal, Dominique
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2004
• Subjects: Animals; Apoptosis - immunology; blood cells; Centrifugation - veterinary; Dog; dogs; Dogs - blood; Dogs - immunology; Elutriation; Female; Flow Cytometry - veterinary; immune response; Immunomagnetic Separation - methods; Immunomagnetic Separation - veterinary; laboratory techniques; Lipopolysaccharide Receptors - immunology; Lymphocyte Culture Test, Mixed - veterinary; Male; Monocytes; Monocytes - cytology; Monocytes - immunology; new methods; purification; PBS; CPM; MLR; Monocytes; FCS; Elutriation; FACS; PBMC; Dog
• ISSN: 0165-2427; 1873-2534
• DOI: 10.1016/j.vetimm.2004.04.012

An elutriation technique was developed to obtain large quantities of pure canine monocytes. Firstly, peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll gradient. Then, the PBMC were separated by an elutriation procedure. We demonstrated that these techniques allow the isolation of canine peripheral blood monocytes with a purity of 64%±7.9 when labelled with anti-CD14 antibody. This purity increased to 83%±2.2 after separation by magnetic anti-CD14 microbeads. The cell viability was more than 95% and apoptotic cells were less than 10%. The monocytes purified by these methods were functionally active in a mixed leukocyte reaction (MLR). A lymphocyte fraction was obtained directly only by elutriation with an average of 79.9%±10.7 of CD5+, 7.9%±3.5 of CD21+ and 1.78%±2.53 of CD14+. Our results indicate that this elutriation procedure is a safe method to purify monocytes as well as lymphocytes, useful in MLR.