Nature - USA (2020-02-13)

4

nature research | reporting summary

April 2018

Animals and other organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals All animals used in this study for BM and imaging analysis are of Mus musculus species, C57/BL6 background strain independent

of genotype and 2-6 months of age. Both males and females were used for all experiments. Mds1-GFP/+, Mds1-GFP/+ Flt3-Cre,

Mds1-CreER/+ Rosa26-CAG-lox-stop-lox-Confetti/+, Rosa26-CAG-lox-stop-lox-tdTomato/+ were generated as detailed described

in methods or purchased from JAX and crossed with our generated strains. A detailed description of the procedure followed to

generate Mds1-GFP/+, Mds1-GFP/+ Flt3-Cre and Mds1-CreER mouse lines is included in the methods section. For Tamoxifen

induction and Cyclophosphamide/GCSF experiments the procedure as well as doses are detailed described in the methods

section.

Wild animals The study did not involve wild animals.

Field-collected samples The study did not involve field-collected samples.

Flow Cytometry

Plots

Confirm that:

The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).

All plots are contour plots with outliers or pseudocolor plots.

A numerical value for number of cells or percentage (with statistics) is provided.

Methodology

Sample preparation Bone marrow isolation was performed using crushing methodology, followed by red blood cell lysis. MACS beads and

quadroMACS (LS columns) were used for lineage depletion or c-kit enrichment. 40um filters were used to ensure single cell

suspension prior FACS analysis. Antibody staining was performed for 45min, on ice, in PBS-2% FBS to ensure good staining and

high viability of the cells. The detailed BM isolation protocol is included in the methods section.

Instrument BD LSR II Flow cytometer was used for flow cytometry analysis, BD FACSAria II was used for cell sorting.

Software BD FACSDIVA Software was used for data collection, FlowJo software (Tree Star) was used for data analysis.

Cell population abundance Cells were sorted with Purity modes at 80-85% efficiency. Post sort fractions analyzed were at least 95% pure. Sorted samples

where double sorted to ensure purity of the sorted populations. In experiments in which low cell numbers of sorted cells was

used, cells where secondary sorted directly in plates to ensure accuracy of cell number.

Gating strategy FSC/SSC preliminary gating was used to exclude debris (lower FSC) and to restrict the main bone marrow population including

larger cells such as granulocytes that are found in higher SSC levels. Back-gating was used to ensure that all excluded populations

were debris or dead cells (positive for DAPI) and that all included populations were part of the various positive antibody

fractions. FSC-H vs. FSC-A was used to exclude doublet cells. SSC-H vs, SSC-A was used as an additional secondary doublet

exclusion step. Dead cells were identified using DAPI staining in all experiments. Gating for negative/positive populations for all

antibodies was performed using a negative control (no stain) followed by single color positive control for each antibody.

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.