Anti cd21

Peripheral blood mononuclear cell (PBMC) isolation was carried out via a differential centrifugation method exactly as described by Kol et al.[45 (link)] PBMCs were harvested and enumerated using an automated cell counter (Coulter ACT diff, Beckman Coulter, Brea, CA). PBMCs were depleted of monocytes, B cells and granulocytes via an LD column (MACS Separation Columns, Miltenyi Biotec, Auburn, CA) using a cocktail of mouse anti-canine antibodies including anti-CD11b (clone CA16.3E10), anti-CD8α (clone CA9.3D3), anti-CD21 (clone CA2.1D6) and goat-anti mouse IgG-microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Flow through cells were collected and treated with an anti-canine CD4 antibody (clone CA13.1E4) followed by goat-anti mouse IgG-microbeads (Miltenyi Biotec) and run on an MS column (MACS Separation Columns, Miltenyi Biotec), according to the manufacturer’s instructions, to yield the final CD4+ T cell enriched fraction. All mouse anti-canine antibodies were purchased from the Leukocytes Antigen Biology Lab, UCD School of Veterinary Medicine.

Immunophenotype was performed on heparinized peripheral blood samples obtained from each patient by means of flow cytometry. 100 μL of blood were stained with two multicolour antibody panels in order to evaluate different lymphocyte subsets, including B cell subpopulations (naïve follicular B cells, marginal zone B cells, switched memory B cells and transitional B cells) and recent thymic emigrants (RTE).
Panel for B cells analysis contained the following antibodies: anti-CD45, anti-CD19, anti-CD38 (Becton Dickinson), anti-CD27, anti-IgM, anti-IgG, anti-CD21, anti-CD10 and anti-IgD (Miltenyi Biotec).
Panel for RTE analysis contained the following antibodies: anti-CD45, anti-CD19, anti-CD4, anti-CD8 (Becton Dickinson), anti-CD3, anti-CD45RA, anti-CD31, anti-CD16 and anti-CD56 (Miltenyi Biotec).
Samples were acquired with MACSQuant Analyzer 10 (Miltenyi Biotec) and analyzed with FlowLogic software (version 7.2.1, Inivai Technologies).