Evaluation of elutriation and magnetic microbead purification of canine monocytes.
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.
de Carvalho
Camila Miranda
CM
Immunology Department, Etablissement Français du Sang de Lyon, 1-3 Rue du Vercors, 69007 Lyon, France. immunologie.lyon@efs.sante.fr
Bonnefont-Rebeix
Catherine
C
Picandet
Stephanie
S
Bernaud
Janine
J
Phothirath
Phoukham
P
Chabanne
Luc
L
Marchal
Thierry
T
Magnol
Jean-Pierre
JP
Rigal
Dominique
D
D000818:Animals; D017209:Apoptosis / Q000276:immunology; D002498:Centrifugation / Q000662:veterinary; D004285:Dogs / Q000097:blood* / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D018189:Immunomagnetic Separation / Q000379:methods / Q000662:veterinary*; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D007959:Lymphocyte Culture Test, Mixed / Q000662:veterinary; D008297:Male; D009000:Monocytes / Q000166:cytology* / Q000276:immunology
D018950:Lipopolysaccharide Receptors