Separation of peripheral lymphocytes by magnetic cell sorting in the dog

Background: The study of pure lymphocyte populations is often frustrating because of difficulties in sorting cells. Usually we rely on gradient separation of peripheral blood mononuclear cells (PBMC), which requires large quantities of blood and does not yield specific subsets. Objective: To achieve sorting of lymphocytes using a small quantity of whole blood. Methods: A MACS separation system (Miltenyi Biotec, Bergisch Gladbach Germany) was used. 300–500 mL of EDTA blood was used directly and after RBC lysis. Positive selection (CD31) and granulocytes1monocytes (CD11b1) depletion were tested. After staining with monoclonal antibodies (MAbs), cells were magnetically labeled with anti-FITC and anti-IgG1 microbeads and passed through MS columns placed in the magnetic field (MACS separator), thus collecting the CD3- or CD11b-negative fraction. Removing the column from the separator, the magnetically retained (CD31and CD11b1) fraction was eluted in a separate tube. Percentages of lymphocytes and granulocytes1monocytes (scatter properties and/or CD3 and CD11b expression) and vitality (PI staining) were recorded by flow cytometry. Results: Whole blood yielded poor results. After RBC lysis, purity of lymphocyte population using anti-FITC microbeads was 85% (recovery 95%) and 21% (recovery 99%) for positive selection and depletion method, respectively. Better results were obtained for positive selection increasing beads/MAb ratio. Anti-IgG1 microbeads returned purity of 92% (recovery 90%) and 87% (recovery 99%) in CD31enrichment and CD11b1depletion, respectively. CD11b1depletion using blood stored 24 hours at 41C yielded lower purity with equal recovery. PI-positive cells were o1% in all cases. Lymphocytes were successfully cultured in all cases. Conclusions: MACS separation system can be used to separate lymphocytes or lymphocyte subpopulations from a small quantity of canine whole blood. Previous RBC lysis is required. Determination of the optimal concentration of MAb respect to microbeads is crucial. The lymphocytes collected are vital and can be cultured.