Lymphoid cells from bone marrow, thymus and spleen were isolated from 8-week old mice. To reveal T cell progenitor subsets in the thymus, single cell suspensions were stained with conjugated antibodies, specific for CD3-FITC (Biolegend), CD4-APC, CD8a-PerCp-Cy5.5, CD25-PE, CD44-APCCy7 (Biolegend), TCRβ-Pacific Blue (Biolegend). To analyse lymphoid subsets in the spleen, single cell suspensions were stained with CD3-FITC, CD4-APC, CD8a-PerCp-Cy5.5, CD19-APCH7, CD45R(B220)-PacificBlue, IgD-PE (eBioscience), IgM-PECy7 (eBioscience). To define the B cell progenitor subsets, suspensions from the bone marrow were stained with IgD-FITC, CD25-PE, IgM-PECy7 (eBioscience), CD45R (B220)-PacificBlue, CD117 (cKit)-APC (eBioscience), CD19-APCH7. Dead cells were excluded from the analysis by propidium iodide staining. Antibodies were purchased from BD Pharmingen unless mentioned otherwise. Samples were measured on a FACS Fortessa® and analysed using FlowJo® software (Version: 10.0.8r1).
Pilzecker B., Buoninfante O.A., Pritchard C., Blomberg O.S., Huijbers I.J., van den Berk P.C, & Jacobs H. (2016). PrimPol prevents APOBEC/AID family mediated DNA mutagenesis. Nucleic Acids Research, 44(10), 4734-4744.
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