Protocol detail

Detailed FACS Sorting and Analysis

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For FACS sorting and analysis, we used previously described staining protocols (Mossadegh-Keller et al., 2013 (link)), published stem and progenitor cell definitions (Bryder et al., 2006 (link)), FACSCanto, LSRII, and FACSAriaIII equipment, and DIVA software (BD), analyzing only populations with at least 200 events. The following antibodies were used for staining cells: anti-CD117 (clone 2B8; BD), anti–Sca-1 (clone D7; BioLegend), anti-CD34 (clone RAM34; BD), anti-CD16/32 (clone 2.4G2; BD), anti-CD11b (clone M1/70; BD), anti-CD19 (clone 1D3; BD), anti-CD3e (clone 145-2C11; BioLegend), anti-Ly6G (clone 1A8; BioLegend), anti-Ly6C (clone HK1.4; BioLegend), anti-CD115 (clone AFS98; eBioscience), anti-CD45.2 (clone 104; BD), anti-CD45.1 (clone A20; BD), anti-B220 (clone RA3-6B2; eBioscience), anti-Ter119 (clone TER-119; eBioscience), and anti-CD71 (clone R17217; eBioscience). LIVE/DEAD Fixable Violet Dead cell dye (Invitrogen) was used as a viability marker. Information describing gating strategies is shown in Figs. S1 and S2.

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Kandalla P.K., Sarrazin S., Molawi K., Berruyer C., Redelberger D., Favel A., Bordi C., de Bentzmann S, & Sieweke M.H. (2016). M-CSF improves protection against bacterial and fungal infections after hematopoietic stem/progenitor cell transplantation. The Journal of Experimental Medicine, 213(11), 2269-2279.

Publication 2016

DIVA software anti–Sca-1 (clone D7; anti-CD34 (clone RAM34; anti-CD16/32 (clone 2.4G2; anti-CD11b (clone M1/70; anti-CD19 (clone 1D3; anti-CD3e (clone 145-2C11; anti-Ly6G (clone 1A8; anti-Ly6C (clone HK1.4; anti-CD115 (clone AFS98; anti-CD45.2 (clone 104; anti-CD45.1 (clone A20; anti-B220 (clone RA3-6B2; anti-Ter119 (clone TER-119; LIVE/DEAD Fixable Violet Dead cell dye

Antibodies Cd11b Cd71 Cell Clone Figs Populations Progenitor cell Sca 1 Stem Violet

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Variable analysis

independent variables

Staining protocols (Mossadegh-Keller et al., 2013)
Stem and progenitor cell definitions (Bryder et al., 2006)
FACS equipment (FACSCanto, LSRII, and FACSAriaIII)

dependent variables

Populations with at least 200 events

control variables

DIVA software (BD)
Antibodies: anti-CD117, anti–Sca-1, anti-CD34, anti-CD16/32, anti-CD11b, anti-CD19, anti-CD3e, anti-Ly6G, anti-Ly6C, anti-CD115, anti-CD45.2, anti-CD45.1, anti-B220, anti-Ter119, and anti-CD71
LIVE/DEAD Fixable Violet Dead cell dye (Invitrogen) as a viability marker

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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