Appendix 1—key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) |
C57Bl/6 | Harlan Labs. 044 |
RRID:MGI:2161078 | C57BL/6NHsd; CD45.2+ |
| Genetic reagent (M. musculus) |
B6.SJLPtprca Pepcb/BoyJ | Jackson Labs 002014 | RRID:MGI:109863 | CD45.1+ |
| Genetic reagent (M. musculus) |
Mx1-Cre | Jackson Labs 003556 |
RRID:MGI:4358794 | Tg(Mx1-cre)1Cgn |
| Genetic reagent (M. musculus) |
Traf6flox/flox | PMID:29386112 | Provided by Dr. Daniel Starczynowski, CCHMC, Cincinnati, OH | |
| Genetic reagent (M. musculus) |
Lyz2Cre | PMID:10621974 | RRID:MGI:1934631 | Mature myeloid cell promoter driving Cre recombinase expression |
| Genetic reagent (M. musculus) |
Vav-Cre | PMID:14975238 | Provided by Dr. Andrew Roberts, WEHI, Melbourne, Australia | |
| Genetic reagent (M. musculus) |
Lyve1eGFP | Jackson Labs 012601 | RRID:MGI:4421655 | Lyve1tm1.1(EGFP/cre)Cys
Enhanced green fluorescent protein (EGFP) in these knock-in mice is driven by the Lyve1 (lymphatic vessel endothelial hyaluronan receptor 1) promoter |
| Genetic reagent (M. musculus) |
β-actin-eGFP | Jackson Labs, 003291 | RRID:MGI:2686773 | B6 ATCTb-EGFP Enhanced GFP (eGFP) cDNA under the control of a chicken β-actin promoter and cytomegalovirus enhancer, have widespread eGFP fluorescence, with the exception of erythrocytes and hair |
| Genetic reagent (M. musculus) |
CX3CR-1GFP | Jackson Labs 005582 |
RRID:MGI:J:84544 | Enhanced green fluorescent protein (EGFP) sequence replacing the first 390 bp of the coding exon (exon 2) of the chemokine (C-X3-C motif) receptor 1 (Cx3cr1) gene |
| Lipopolysaccharide | LPS | Sigma-Aldrich | Cat# L2630 | Biological source is Escherichia coli (O111:B4)
Purified by phenol extraction Stimulation with this generates various pro inflammatory cytokines |
| Enzyme | Collagenase-II | Thermo Fisher Scientific, Gibco | Cat# 17101015 | Biological source is Clostridium histolyticum, digestion of bone, cartilage, tissues |
| Enzyme | Dispase | Thermo Fisher Scientific, Gibco. | Cat# 17105041 | Biological source is Bacillus polymyxa
Helpful in dissociation of primary cells from the tissues |
| Lysing buffer | Pharm Lyse (red blood lysing buffer) |
BD Biosciences | Cat# 555899 | Helpful in red blood cell lysis and results in good light scatter separation of lymphocytes and red blood cell debris when used in flow cytometry |
| Methylcellulose media | Methocult-GF M3434 | StemCell Technology | Cat# 03434 | Semisolid medium to growth murine HPCs |
| Base methylcellulose medium | Methocult-M3134 | StemCell Technology | Cat# 03134 | Incomplete medium that contains 2.6% methylcellulose in Iscove's MDM |
| Cytokine | Recombinant murine GM-CSF |
PeproTech | Cat# 315-03 | Biological source is E. coli
Hematopoietic growth factor stimulates macrophages and neutrophils Produced in endothelial cells, fibroblasts |
| Cytokine | Recombinant murine G-CSF |
PeproTech | Cat# 250-05 | Biological source is E. coli
Hematopoietic growth factor that stimulates the committed progenitor to neutrophils and improve the functional activities |
| Cytokine | Recombinant murine M-CSF | PeproTech | Cat# 315-02 | Biological source is E. coli
It facilitates the hematopoietic recovery after the bone marrow transplantation |
| Cytokine | Human G-CSF | NEUPOGEN (filgrastim, Amgen) |
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| Lineage cocktail antibody | Anti-CD3e (CD3ε chain), anti-TER-119/erythroid cells (Ly-76), anti-Gr1 (Ly6G and Ly-6C), anti-CD45R (B220), anti-CD11b (integrin α chain, Mac1α) | Pharmingen, BD Biosciences |
559971 RRID:AB_10053179 |
Sorting out or depleting lineage expressing cells and enriching hematopoietic progenitors in bone marrow CD3e is biotin Hamster anti-mouse, dilution 1:200, CD45R is rat anti-mouse dilution 1:200, CD11b is rat anti-mouse, Ter119 is rat anti-mouse |
| Recombinant protein | Cy7-APC-Cy7-conjugated streptavidin | Pharmingen, BD Biosciences |
554063 RRID:AB_10054651 |
|
| Antibody | APC-conjugated anti-c-Kit | Pharmingen, BD Biosciences |
553356 RRID:AB_398536 |
Clone 2B8 Rat anti-mouse specifically binds to C-kit a transmembrane tyrosine kinase receptor Dilution 1:200 |
| Antibody | PECy7-conjugated anti-Sca1 | Pharmingen, BD Biosciences |
558162 RRID:AB_647253 |
Clone D7 Rat anti-mouse Dilution 1:200 |
| Antibody | eFluor 450-conjugated anti-CD34 | Affymetrix, eBioscience | RRID:AB_2043838 | Clone RAM34 Monoclonal antibody reacts with mouse CD34 Dilution 1:200 |
| Antibody | PerCP Cy5.5-conjugated anti-Fcγ-RII/III | Pharmingen, BD Biosciences |
560540 RRID:AB_1645259 |
Clone 2.4G2 It specifically recognizes a common non-polymorphic epitope on the extracellular domains of the mouse FcrγIII Rat anti-mouse Dilution 1:200 |
| Antibody | FITC-conjugated anti-CD45.1 | Pharmingen, BD Biosciences |
553775 RRID:AB_395043 |
Clone A20 Monoclonal antibody specifically binds to CD45.1 of all leukocytes Dilution 1:200 |
| Antibody | PECy7-conjugated anti-CD45.2 | Pharmingen, BD Biosciences |
560696 RRID:AB_1727494 |
Clone 104 This recognizes CD45 on all leukocytes that of most mouse strains example: C57BL6, Balb/C, etc. Mouse monoclonal Dilution 1:200 |
| Antibody | APC-conjugated anti-CD11b | Pharmingen, BD Biosciences |
Cat# 553312 RRID:AB_398535 |
Clone M1/7 Specifically binds to CD11b or integrin alfa M Rat anti-mouse Dilution 1:200 |
| Antibody | -APC-Cy7-conjugated anti-B220 (clone RA3-6B2 | Pharmingen, BD Biosciences |
552094 RRID:AB_394335 |
Clone RA3-6B2 Binds to an extracellular domain of the transmembrane CD45, which is expressed in all B lymphocytes Rat anti-mouse Dilution 1:200 |
| Antibody | PE-conjugated anti-CD3e | Pharmingen, BD Biosciences |
552774 RRID:AB_394460 |
Clone 145-2 C11 Binds to T-cell receptor expressed CD3 complex that is expressed in thymocytes and mature T cells, etc. Hamster anti-mouse Dilution 1:200 |
| Antibody | BD Horizon V450-conjugated anti-Gr1 | Pharmingen, BD Biosciences |
560454 RRID:AB_1645285 | RB6-8C5 Rat anti-mouse, clonality unknown Dilution 1:200 |
| Antibody | PerCP efluor710 anti-CD115 | Thermo Fisher Scientific, eBioscience | 46-1152-82 RRID:AB10597740 |
Clone AFS98 Monoclonal antibody against mouse Cd115, receptor of macrophage colony-stimulating factor Dilution 1:200 |
| Antibody | R-phycoerythrin conjugated anti-CD135 | BD Biosciences | Cat# 562537 RRID:AB_2737639 |
A2F10.1 This is a monoclonal antibody specifically binds to Flk2-Flt3 Dilution 1:200 |
| DNA die | Hoechst 33342 | Fisher Scientific, Invitrogen | Cat# H3570 | This is a cell permeant nuclear counterstain that emits blue florescence when bound to dsDNA 10 mg/mL |
| Antibody | Rabbit non-conjugated monoclonal anti-phospho-SNAP23(Ser95) | Paul A. Roche | Center for Cancer Research, National Cancer Institute, NIH | |
| Fixation buffer | BD Cytofix | BD Biosciences | Cat# 554655 | For immunofluorescent staining of intracellular cytokines |
| Fixation/permeabilization Kit |
BD Cytofix/Cytoperm | BD Biosciences | Cat# 554714 | This provides fixation and permeabilization of the cells |
| Perm/Wash buffer | BD Perm/Wash | BD Biosciences | Cat# 554723 | This is used in intracellular cytokine staining to permeabilize cells and can be used as antibody diluent |
| Antibody | Secondary Alexa Fluor 488-conjugated, goat anti-rabbit | Thermo Fisher Scientific, | RRID:AB_2633280 | Used as secondary antibody Goat polyclonal Dilution 1:500 |
| Software, algorithm | FlowJo xV0.7 | https://www.flowjo.com/solutions/flowjo | RRID:SCR_008520 | |
| In Situ Cell Death Detection Kit | TMR red | Sigma-Aldrich, Roche |
Cat# 12156792910 | This is used to detect apoptosis on a single-cell level |
| Depletion kit | Lineage Cell Depletion | Miltenyi Biotech | Cat# 130-090-858 | Depletion of mature hematopoietic cells |
| Plasma membrane dye | Dil stain | Thermo Fisher Scientific, Invitrogen | Cat# D3911 | This is a lipophilic membrane stain that diffuses laterally to stain the entire cell |
| Detergent solution |
Triton X-100 | Sigma-Aldrich | Cat# X100 | A mild detergent used to break the cell membrane |
| Antibody | Anti-GFP | Abcam | Cat# ab13970 RRID:AB_300798 |
Chicken polyclonal antibody Dilution 1:500 |
| Antibody | Purified rat anti-mouse panendothelial cell antigen | 550563 RRID:AB_393754 |
Clone Meca-32 Mouse monoclonal Dilution 1:500 |
|
| Antibody | Secondary antibodies goat anti-rat Alexa Fluor-488 |
Thermo Fisher Scientific, Invitrogen | Cat# A-11006 RRID:AB_2534074 |
Goat anti-rat Clonality unknown Dilution 1:500 |
| Antibody | Secondary antibodies goat anti-chicken Alexa Fluor-568 | Thermo Fisher Scientific, Invitrogen | Cat# A-11041 RRID:AB_2534098 |
Goat anti-chicken Clonality unknown Dilution 1:500 |
| Software | Imaris | http://www.bitplane.com/imaris/imaris | RRID:SCR_007370 | |
| Antibody | Anti-mouse CD62L | BioXcell | Cat# BE0021 RRID:AB_1107665 |
This antibody reacts with L-selectin that is expressed by neutrophils, monocytes, and the majority of T- and B cells- Mouse monoclonal Dilution 1:500 |
| Antibody | Anti-CD19 | BD Biosciences | Cat# 552854 RRID:AB_394495 |
Rat monoclonal antibody Dilution 1:200 |
| Enzyme-free cell dissociation buffer | Cell dissociation buffer | Thermo Fisher–Gibco | Cat# 13150016 | Dissociation of mammalian cells |
| Recombinant human fibronectin fragment | RetroNectin | Takara | Cat# T202 | This reagent promotes co-localization of lenti or retrovirus with the target cells and promotes the transduction efficiency |
| Antibody | Anti-CD169 | eBioscience | Cat# 12-5755-82 RRID:AB_2572625 |
This antibody specifically binds to CD169, the receptor expressed in subset of macrophages and plays an important role in cell-cell adhesion |
| Antibody | Anti-F4/80 | Thermo Fisher Scientific, eBioscience | Cat# 48-4801-82 RRID:AB_1548747 |
Rat anti-mouse Dilution 1:200 |
| Protease inhibitor cocktail | Complete Protease inhibitor cocktail | Sigma-Aldrich, Roche |
Cat# 11697498001 | Inhibits broad spectrum of proteases |
| ELISA | Mouse Ccl19/MIP-3 | R&D System | Cat# DY440 | |
| ELISA | Mouse Ccl21/6Kine | R&D System | Cat# AF457 | |
| Recombinant protein | Mouse anti-Ccr7 | R&D System | Cat# MAB3477 | |
| Recombinant protein | Mouse anti-Ccl19 | R&D System | Cat# 440-M3 | |
| Inhibitor | Ubiquitin-conjugating enzyme E2 N (UBE2N) inhibitor or Ubc13 | Sigma-Aldrich | Cat# 662107 | This inhibitor controls the biological activity of UbcH13 |
| Inhibitor | IRAK1 inhibitor | Sigma-Aldrich | Cat# I5409 | |
| Inhibitor | PS1145 dihydrochloride | Sigma-Aldrich | Cat# P6624 | IκB kinase inhibitor |
| Inhibitor | Monensin Solution 100× | Thermo Fisher Scientific, eBioscience | Cat# 00-4505-51 | Inhibitor of intracellular transport |
| Assay | TUNEL-TMR | Roche | Cat# 12156792910 | Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL ) assay to detect apoptotic cells labeled with tetra-methyl-rhodamine |