Comprehensive Immunophenotyping of Murine Hematopoietic Cells

Blood, BM cells and splenocytes were collected as previously described^{19 (link),40 (link)}. The different cell samples (0.5–2 × 10⁶ cells) were stained for the indicated surface markers (see Supplemental Table S2 online) for 20 min at room temperature and subsequently washed twice with buffer A. We distinguished live and dead cells by adding SYTOX Green (S7020, Life Technologies) or DAPI (D9542, Sigma-Aldrich) 5 min before FACS analysis. Haematopoietic progenitors were isolated from the spleen and BM by magnetic-activated cell sorting (MACS) using a Lineage Cell Depletion Kit (130-090-858, Miltenyi) and MACS separation MS columns (130-042-201, Miltenyi) prior staining. Unstained cells were used as a negative control to establish the flow cytometer voltage settings, and single-colour positive controls were used to adjust compensation. The absolute number of cells was calculated by adding Perfect-Count Microspheres (CYT-PCM-100, Cytognos) to the flow cytometry samples. Apoptosis, cell cycle, and cell proliferation were determined by flow cytometry as previously described^{19 (link),40 (link)}. The flow cytometry data were acquired using a FACSCanto II and analysed with FACSDiva (Becton and Dickinson) or FlowJo software.

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Sánchez Á., Orizaola M.C., Rodríguez-Muñoz D., Aranda A., Castrillo A, & Alemany S. (2020). Stress erythropoiesis in atherogenic mice. Scientific Reports, 10, 18469.

Publication 2020

SYTOX Green DAPI Lineage Cell Depletion Kit MACS separation MS columns Perfect-Count Microspheres (CYT-PCM-100, FACSCanto II FACSDiva

Apoptosis Blood Buffer Cell cycle Cell proliferation Cells Dapi Flow cytometry Haematopoietic Microspheres Spleen Sytox green

Corresponding Organization :

Other organizations : Instituto de Investigaciones Biomédicas Sols-Morreale, Autonomous University of Madrid, Consejo Superior de Investigaciones Científicas

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Variable analysis

independent variables

Cell type (blood, BM cells, and splenocytes)

dependent variables

Cell surface marker expression
Cell viability (live and dead cells)
Apoptosis
Cell cycle
Cell proliferation

control variables

Cell number (0.5–2 × 10^6 cells)
Staining duration (20 min)
Staining temperature (room temperature)
Washing procedure (twice with buffer A)
Flow cytometer voltage settings (using unstained cells as negative control)
Compensation (using single-colour positive controls)
Absolute cell number calculation (using Perfect-Count Microspheres)

positive controls

Single-colour positive controls for adjusting compensation

negative controls

Unstained cells for establishing flow cytometer voltage settings

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