Flow cytometry analyses_bone marrow stem and progenitor cells, spleen T, B and myeloid cells

The analysis of the effects of miR-221/222 gene cluster on unperturbed, short-term social stress perturbed and transplantation-perturbed hematopoiesis in bone marrow and in peripheral lymphoid tissues of mice.
Conclusion:
We compare pool sizes and single-cell transcriptomes of HSC and MPPs in unperturbed or stress-perturbed, miR-221/222-proficient or deficient states. MiR-221/222-deficiency in hematopoietic cells was induced in C57BL/6J mice by conditional vav-cre-mediated deletion of the floxed miR-221/222 gene cluster. Social stress as well as miR-221/222-deficiency, alone or in combination, reduced HSC pools 3-fold, increased MPPs 1.5-fold. It also enhanced granulopoisis in the spleen. Furthermore, combined stress and miR-221/222-deficiency increased the erythroid/myeloid/granulocytic precursor pools in bone marrow. Finally, stress by serial transplantations of miR-221/222-deficient HSC selectively exhausted their lymphoid differentiation capacities, while retaining their ability to home to BM and to differentiate to granulocytes. The BD Fortessa flow cytometer was quality controlled daily by the staff of the flow cytometry core facility of the Institute. All reagents were titrated over a broad range of concentrations to ensure maximal performance. Internal controls were used as described in the online material. Briefly: Unstained and single stained controls were used for pre-experimental setup and for compensation. The flow cytometer was set up before experiments using nine-color beads (BD FACS, BD Biosciences, San Jose, CA, USA). Voltages were adjusted such that fluorescence intensity was identical for each antibody over the experiments.