Supplementary Material and Methods s1

SUPPLEMENTARY MATERIAL AND METHODS

Hepatic function: Serum AST and ALT levels were used to determined hepatic function. Blood was collected by heart puncture. All samples were analyzed using colorimetric assays, using commercial kits to detect TGO and TGP (both from Labtest, Lagoa Santa, Brazil).

Immunolabeling The following monoclonal antibodies (mAbs) were used to define the different populations of hematopoietic cells: Ter-119 (Ly-76), Mac-1 (myM1/70), Gr-1 (RB6-8C5), B220 (RA3-6B2), CD3 (145-2C11), c-Kit (2B8), Sca-1 (Ly-6A/E-D7), NK1.1 (PK136), CD49 (DX5), CD90.1 (Thy1.1-HIS51) F4/80 (6F12) and FLK-2 (A2F10). To identify mature populations, whole bone marrow cells (106/sample) were labeled with the follows antibodies: Gr-1- PE, Mac-1-PE/Cy7, F4/80-FITC, NK1.1-FITC and CD49-PE/Cy7. Cells were incubated with the antibodies for 20 min and then washed. To identify primitive hematopoietic populations, whole bone marrow cells (3x106cells/sample) were labeled with a mature lineage (Lin) antibody cocktail (Gr-1-PE, Mac-1-PE, CD3e-PE, Ter-119-PE and B220-PE), CD90.1 (Thy1.1.)-FITC, FLK-2-PE, Sca-1- PE/Cy7 and c-Kit-APC for 20 min. The cells were then washed. Mac-1+Gr-1+ cells were defined as myeloid cells. Mac-1+F4/80+ cells were defined as monocytes cells. NK1.1+CD49+ cells were defined as natural killer cells. HSCs were defined as Lin-FLK-2-Sca-1+c-Kit+Thy1.1low [18]. Hematopoietic progenitors were defined as Lin-FLK-2-Sca-1-c-Kit+Thy1.1low [18]. All antibodies were purchased from Becton Dickinson (San Jose, California, USA). All cytometry analysis in this study was performed on a FACSCalibur (Becton Dickinson, USA) flow cytometer using Cell Quest (Becton Dickinson, USA) and FlowJo software (Tree Star Inc., USA).

Cellular Evaluation The leukocytes (103cells/mm3) count determination was performed using an automated cell counter (ABX ABC Vet Hematology Analyzer, HORIBA).

Differential leukocyte The count was performed on blood smears May-Grunwald Giemsa solutions (Sigma Chemical Co., St Louis, MO, USA).

18. Barbosa CM, Leon CM, Nogueira-Pedro A, Wasinsk F, Araujo RC, Miranda A, Ferreira AT, Paredes-Gamero EJ. Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines. Cell Death Dis 2011; 2: e165.