Essay On Cell Transplantation

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HSC and FC were isolated from bone marrow of mouse by multiparameter, live sterile cell sorting (FACSVantage SE and FACSAria; Becton Dickinson, Mountainview, CA), as previously described.2 Briefly, bone marrow cells was isolated and resuspended in a single cell suspension at a concentration of 100 x 106 cells/ml in sterile cell sort media (CSM), containing sterile 1 x Hank’s Balanced Salt Solution without phenol red (GIBCO, Grand Island, NY), 2% heat-inactivated fetal calf serum (FCS; GIBCO), 10 mM HEPES buffer (GIBCO), and 50 g/ml Gentamicin (GIBCO). Directly labeled mAb were added at saturating concentrations and the cells were incubated for 30 min on ice and washed twice using CSM. Cells were resuspended in CSM at 2.5 x 106…show more content…
The peripheral blood, thymus, spleen, and bone marrow were harvested and cells staining with CD4 FITC (GK1.5; Rat IgG2a), CD25 APC (PC61; Rat IgG1), Foxp3 Pacific Blue (FJK-16s; Rat IgG2a), CCR5 PE (HM-CCR5-7A4; Armenian hamster IgG) and CXCR3 PerCP Cy5.5 (CXCR3-173; Armenian hamster IgG). CD4+CD25+Foxp3+, CD4+CD25+Foxp3+CXCR3+, and CD4+CD25+Foxp3+CCR5+ Treg were analyzed by flow cytometry. FC morphology. Wright-Giemsa staining was performed on cytospins of 100,000 FC from B6 or Flt3-L-KO B6 mice after being fixed in methanol. Slides were examined for dendritic morphology under optical microscopy. Evaluation of donor chimerism. Donor engraftment was evaluated by peripheral blood lymphocyte (PBL) typing using 4-color flow cytometry as previously described.2 Briefly, whole blood from mouse recipients was collected in heparinized tubes, and aliquots of 100 μl were stained with donor-specific anti-H-2Kb FITC (AF6-88.5; mouse IgG2a) along with a combination of the following mAbs (PharMingen): CD8α PerCP (53-6.7; rat IgG2a), CD4 PerCP (RM4-5; rat IgG2a), β-TCR APC (H57-597; Armenian hamster IgG), B220 PerCP, CD11b APC, NK1.1 PE (PK136; mouse IgG2a), Pan-NK cell PE, CD11c PE, Gr-1 PE (RB6-8C5; rat IgG2b) mAbs. Statistical analysis. Experimental data were presented as the mean plus or minus SEM. Statistical significance was assessed using the student’s t-test; P < 0.05 was considered significant. Graft survival was calculated according to the
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