Flow cytometry analysis

To study cell marker expression, the following fluorochrome-conjugated antibodies were used as described [4, 16]: anti-CD45 (leukocyte marker; clone 30-F11; eBioscience, San Diego, CA, USA), anti-CD3 (T lymphocyte marker; clone 17A2; BD Biosciences, Mississauga, ON, Canada), anti-B220 (B lymphocyte marker; clone RM4-5; BD Biosciences), anti-CD4 (helper T lymphocyte marker; clone GK1.5; eBioscience), anti-CD8b (cytotoxic T lymphocyte marker; clone eBioH35-17.2; eBioscience), anti-CD25 (regulatory T lymphocyte marker; clone PC61.5; eBioscience), anti-Foxp3 (regulatory T lymphocyte marker; clone FJK-16 s; eBioscience), anti-F4/80 (macrophage marker; clone BM8; eBioscience), anti-CD11b (macrophage and granulocyte marker; clone M1/70; eBioscience), anti-Gr1 (granulocyte marker; Ly-6G, Clone RB6-8C5; eBioscience), or relevant isotypic controls in PBS-1%BSA. We used 7-amino-actinomycin D to assess cell viability (eBioscience). Cells were acquired and analyzed using a CyFlow ML cytometer (Partec North America, Inc., Swedesboro, NJ, USA) and FCS Express software (De Novo Software, Los Angeles, CA, USA).

Hematopoietic stem cells were quantified in bone marrow with the Mouse Hematopoietic Stem Cell Isolation Kit (BD Biosciences). It includes a lineage cocktail (lin), which contains antibodies against CD3, CD11b, B220, Ly6C, Gr1, and TER-119 to exclude differentiated cells. Both long-term reconstituting (LTR) and STR hematopoietic stem cells express the cell markers Sca-1 and c-Kit, whereas only STR expresses CD34. Therefore, lin⁻/Sca-1⁺/cKit⁺/CD34⁻ cells are LTR and lin⁻/Sca-1⁺/cKit⁺/CD34⁺ cells are designated STR.