(A) Anatomy of lumbar spinal cord and DRGs connected to the sciatic nerve. The location of the crush site within the nerve trunk and the tissue segment collected for flow cytometry (red bracket) are …
Cells were first gated with forward scatter (FSC-A) and side scatter (SSC-A) to exclude debris. Cells were then gated with forward scatter height (FSC-H) and FSC-A to find single cells and to …
(A) Anatomy of lumbar spinal cord and DRGs connected to the sciatic nerve. The location of the crush site within the nerve trunk and the tissue segment collected for flow cytometry (red bracket) are …
(A) Representative images of L4 DRG cross sections from naïve mice, d1, d3, and d7 post-SNC. Macrophages were stained with anti-Iba1 and anti-F4/80. Neurons were stained with anti-NFH. Scale bar, 50 …
Representative images of L4 DRG cross sections from naïve mice, d1, d3, and d7 post-SNC. T cells were labeled with anti-CD3 (red), neurons with anti-NF200 (white). DAPI (blue) was used for nuclear …
(A) Parbiosis complex of a wildtype (WT) and a tdTomato (tdTom) mouse. Mice were surgically paired at postnatal day 56. The timeline of the experiment is shown. (B) Flow cytometric analysis of …
(A) Cells were first gated with forward scatter (FSC-A) and side scatter (SSC-A) to exclude debris. Cells were then gated with forward scatter height (FSC-H) and FSC-A to find single cells and to …
Analysis of bulk RNAseq data from naïve and axotomized DRGs. DRGs were harvested from sham operated mice, d1, d3, and d7 post-SNC. (A) Network analysis of whole transcriptomes from naive and …
(A) Heatmap of the correlation of WGCNA modules with indicated experimental conditions (sham operated mice and following nerve injury). The values in each cell are Pearson's correlation co-efficient …
(A) Singe-cell transcriptome of injured mouse sciatic nerve at d3 post-SNC, n = 3 biological replicates. Unsupervised Seurat-based clustering identifies 24 cell clusters. Cell type identity for each …
List of top 100 cluster enriched genes for all cell clusters identified in the 3d post-SNC nerve.
First column (P_val), probability of getting the ‘elevated’ expression values in these cells under the null hypothesis that all cells have the same expression of the gene. Second column (avg_logFC), average log2 Fold-Change between cells in this cluster relative to cells in all other clusters. Third column (pct.1), percent of the cluster's cells which express the gene. Fourth column (pct.2), percent of non-cluster cells which express the gene. Fifths column (p_val_adj), p_val adjusted so that 5% of the list is expected to have false positives. Links to STRING REACTOME pathway analysis for each cell cluster are included on the last page.
(A) Dotplot analysis of extracellular matrix molecules prominently expressed in the d3 post-SNC nerve. (B) Dotplot analysis of gene products implicated in axon growth, guidance, and regeneration in …
Heatmap of top genes enriched in perineural mesenchymal cells (pMES), endoneurial mesenchymal cells (eMES), differentiating mesenchymal cells (dMES), and epineural fibroblasts (Fb) in the d3 …
Heatmap of top genes enriched in repair Schwann cell clusters (SC1, SC2, and SC3) in the d3 post-SNC nerve. Expression levels are calibrated to median gene expression. STRING REACTOME pathways are …
Heatmap of top genes enriched in endothelial cell clusters (EC1, EC2, and EC3) in the d3 post-SNC nerve. Expression levels are calibrated to median gene expression. STRING KEGG Pathway analysis and …
Heatmap of top genes enriched in pericyte clusters (PC1 and PC2) in the d3 post-SNC nerve. Expression levels are calibrated to median gene expression. STRING REACTOME Pathways are listed. FDRs …
Dotplot analysis of gene products with immune modulatory function in the d3 post-SNC nerve. Expression levels, normalized to average gene expression (color coded). For each cell cluster, the …
(A–E) Feature plots of Adgre1 (F4/80), Aif1 (Iba1), Cd68 (Scavenger receptor class D), Cx3cr1 (Fractalkine receptor), and CD209a (DC-SIGN) expression in the d3 post-SNC nerve. (F) scRNAseq dot plot …
Heatmap of top genes enriched in monocytes (Mo), macrophage clusters 1–5 (Mac1-5), MoDC, and cDC in the d3 post-SNC nerve. Expression levels are calibrated to median gene expression. STRING KEGG …
(A) Feature plots of Stmn1 (Stathmin-1) and (B) Top2a (DNA topoisomerase II alpha) highlight proliferating cells, including Mac5 cells and a small group of myeloid cells located between clusters …
Pseudo time trajectory analysis of cell differentiation in the d3 post-SNC nerve (A–D) Predicted cell differentiation in the myeloid cell compartment. Slingshot analysis was used to predict how Mo …
Flow cytometric analysis of Mo/Mac in naive and axotomized DRGs. The surface distribution of Ly6C on Mo/Mac (CD45+, CD11b+, Ly6G-, CD11c-) is shown. (A) The percentile of Ly6C cells and (B) the …
(A) Longitudinal sections of naïve and injured (3d and 7d) sciatic nerves from Arg1-YFP reporter mice. The injury site is underlined with a dotted line. Proximal is to the left. Distribution of …
Feature plots of gene products implicated in cholesterol transport and lowering of cellular cholesterol levels. (A) Abca1/CERP (ATP-binding cassette subfamily A1). (B) Abcg1 (ATP-binding cassette …
(A) Cartoon of phagocyte with actin rich phagocytic cup eating a tdTom+apoptotic cell (AC). ‘Eat me’ signals displayed on the surface of AC allow direct or indirect recognition via engulfment …
(A–F) Feature plots and (G–L) Violin plots of Axl (TAM receptor tyrosine kinase AXL), Mertk (TAM receptor tyrosine kinase MER), Lrp1 (Low-density lipoprotein receptor-related protein 1), Pirb …
(A) Dotplot showing expression of engulfment receptors in in naïve Mac in comparison to Mo and Mac1-5 in injured nerve. (B). Dotplot showing expression of ‘don’t eat me’ (Cd47 and Sirpa) signals, as …
(A) Flow cytometric analysis of 3d sciatic nerve from non-parabiotic (tdTom single) mice. Dot plot shows that 96.8% of MoDC (CD11b+, CD11c+, Ly6G-) are tdTom+(B) Flow cytometric analysis of 3d …
(A) Schematic showing conditioning lesion to the sciatic nerve (1) followed by dorsal column lesion (2) and tracer injection in the nerve (3). Experimental time line of conditioning lesion (CL), …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Neurofilament heavy chain (chicken polyclonal) | Aves Lab | NFH | 1:750 |
Antibody | Anti-chicken Cy3 (donkey polyclonal) | Jackson Immunoresearch | 703-165-155 | 1:200 |
Antibody | Iba1 (rabbit polyclonal) | Wako Chemicals | 019–19741 | 1:500 |
Antibody | F4/80 (Rat IgG2b monoclonal) | Thermo Fisher Scientific | ma1-91124 | 1:500 - 1:1000 |
Antibody | CD68 (rabbit polyclonal) | Abcam | ab125212 | 1:500 |
Antibody | GFAP (rabbit polyclonal) | DAKO | Z033429-2 | 1:500 |
Antibody | SCG10 (rabbit polyclonal) | Novus Biologicals | NBP149461 | 1:500 - 1:1000 |
Antibody | CTB (goat polyclonal) | List Biological Laboratories | 703 | 1:10,000 |
Antibody | CD11b (rabbit monoclonal) | Abcam | ab133357 | 1:200-1:1000 |
Antibody | ERK1/2 (rabbit polyclonal) | Cell Signaling | 9102 | 1:5000 |
Antibody | Anti-rabbit HRP (donkey polyclonal) | EMD Millipore | AP182P | 1:2000-1:10000 |
Antibody | CD16/32 (Rat-IgG2a monoclonal) | BD Pharmingen | 553141 | 1 µg / 1 million cells / 25 µL |
Antibody | CD11b-PE-Cy7 (Rat-IgG2b monoclonal) | Thermo Fisher Scientific | 25-0112-82 | 1:200 |
Antibody | Isotype Control-PE-Cy7 (Rat-IgG2b monoclonal) | Thermo Fisher Scientific | 25-4031-82 | 1:100 |
Antibody | CD45-e450 (Rat-IgG2b monoclonal) | Thermo Fisher Scientific | 48-0451-82 | 1:100 |
Antibody | Isotype Control-e450 (Rat-IgG2b monoclonal) | Thermo Fisher Scientific | 48-4031-82 | 1:100 |
Antibody | CD45.1-e450 (Mouse-IgG2a monoclonal) | Biolegend | 110721 | 1:100 |
Antibody | Isotype Control-e450 (Mouse-IgG2a monoclonal) | Biolegend | 400235 | 1:100 |
Antibody | CD45.2-APC (Mouse-IgG2a monoclonal) | Biolegend | 109813 | 1:100 |
Antibody | Isotype Control-APC (Mouse-IgG2a monoclonal) | Biolegend | 400221 | 1:100 |
Antibody | Ly6G-APC-Cy7 (Rat-IgG2a monoclonal) | BD Biosciences | 560600 | 1:100 |
Antibody | Isotype Control-APC-Cy7 (Rat-IgG2a monoclonal) | BD Biosciences | 552770 | 1:100 |
Antibody | CD11c-PerCP-Cy5.5 (ArmHam-IgG monoclonal) | Thermo Fisher Scientific | 45-0114-82 | 1:100 |
Antibody | Isotype Control-PerCP-Cy5.5 (ArmHam-IgG monoclonal) | Thermo Fisher Scientific | 45-4888-80 | 1:100 |
Antibody | Ly6C-FITC (Rat-IgM monoclonal) | BD Biosciences | 561085 | 1:100 |
Antibody | Isotype Control-FITC (Rat-IgM monoclonal) | BD Biosciences | 553942 | 1:100 |
Antibody | Iba1 (goat polyclonal) | Novus Biologicals | NB100-1028 | 1:200 |
Antibody | Anti-goat Alexa Fluor 488 (donkey polyclonal) | Jackson Immunoresearch | 705-545-147 | 1:200 |
Chemical Compound | TOPRO pan-nuclear stain | Thermo Fisher Scientific | T3605 | |
Chemical Compound | Fixable Viability Dye | Thermo Fisher Scientific | 65086614 | 1:500 |
Chemical Compound | Proteinase K | New England Biolabs | P8107S | |
Chemical Compound | 10 mM dNTP mix | Promega | C1141 | |
Chemical Compound | 5X Green GoTaq Buffer | Promega | M791A | |
Chemical Compound | GoTaq DNA polymerase | Promega | M3005 | |
Chemical Compound | Buprenorphine | Par Pharmaceutical | NDC12496-0757-1 | |
Chemical Compound | Ketamine | Par Pharmaceutical | NDC42023-115-10 | |
Chemical Compound | Xylazine | Akorn | NDC59399-110-20 | |
Chemical Compound | Isoflurane | McKesson Corporation | 667940172 | |
Chemical Compound | Rhodamine-conjugated dextran MW 3,000 (Microruby) | Life Technologies | D-7162 | |
Chemical Compound | Cholera toxin B (CTB) | Life Technologies | C34775 | |
Chemical Compound | Puralube Eye ointment | Dechra | NDC-17033-211-38 | |
Chemical Compound | N2 | Gibco | 17502048 | |
Chemical Compound | N1 | Sigma | N6530 | |
Chemical Compound | Leibovitz-15 (L-15) | Gibco | 21083–027 | |
Chemical Compound | Penicillin/Streptomycin | Life Technologies | 15140–122 | |
Chemical Compound | DMEM Ham’s F-12 | Gibco | 10565–018 | |
Chemical Compound | Fetal Bovine Serum | Atlanta Biologicals | S11550 | |
Chemical Compound | Cytosine arabinoside | Sigma-Aldrich | C1768 | |
Chemical Compound | Collagenase type 2 | Worthington Biochemical | LS004176 | |
Chemical Compound | PBS without Calcium, Magnesium | Gibco | 10010023 | |
Chemical Compound | Poly-L-lysine MW 70,000–150,000 | Sigma-Aldrich | P4707 | |
Chemical Compound | laminin | Sigma-Aldrich | L2020 | |
Chemical Compound | Paraformaldehyde | Sigma-Aldrich | 158127–500G | |
Chemical Compound | Triton-X100 | Sigma-Aldrich | T8787 | |
Chemical Compound | Bovine Serum Albumin (BSA) heat shock fraction V | Fisher Scientific | BP1600 | |
Chemical Compound | Hoechst 33342 | Invitrogen | H3570 | |
Chemical Compound | Tissue-Tek O.C.T. Compound | Electron Microscopy Sciences | 62550–01 | |
Chemical Compound | β-Glycerophosphate | Sigma-Aldrich | G9422-100G | |
Chemical Compound | Sodium Orthovanadate (Na3VO4) | Sigma-Aldrich | S6508-10G | |
Chemical Compound | Protease inhibitor cocktail | Sigma-Aldrich | P8340-5ML | |
Chemical Compound | DC Protein Assay Kit | Bio-Rad | 5000111 | |
Chemical Compound | 2x Laemmli sample buffer | Bio-Rad | 1610737 | |
Chemical Compound | β-Mercaptoethanol | EMD Millipore | 6010 | |
Chemical Compound | Blotting-grade blocker | Bio-Rad | 1706404 | |
Chemical Compound | SuperSignal West Pico PLUS Chemiluminescent Substrate | Thermo Fisher Scientific | 34580 | |
Chemical Compound | SuperSignal West Femto Maximum Sensitivity Substrate | Thermo Fisher Scientific | 34095 | |
Chemical Compound | WesternSure PREMIUM Chemi Substrate | Li-Cor Biosciences | 926–95000 | |
Chemical Compound | Fixable Viability Dye eF506 | Thermo Fisher Scientific | 65-0866-14 | |
Chemical Compound | TRIzol | Thermo Fisher Scientific | 15596026 | |
Chemical Compound | Dispase | Sigma-Aldrich | D4693 | |
Chemical Compound | Actinomycin D | Sigma-Aldrich | A1410 | |
Chemical Compound | Percoll | Sigma-Aldrich | P4937 | |
Chemical Compound | MACS buffer | Miltenyi | 130-091-376 | |
Chemical Compound | Hanks balanced salt solution | Gibco | 14025092 | |
Chemical Compound | Sucrose | Fisher Scientific | S5-500 | |
Other | Pasteur pipette | VWR | 14673–010 | |
Other | Flat Bottom Cell Culture Plates, 24-well | Corning | 3524 | |
Other | Superfrost Plus Microscope Slides | FisherScientific | 12-550-15 | |
Other | Zeiss Axio Observer Z1 | Zeiss | 491912-0049-000 | |
Other | Zeiss Axiocam 503 mono camera | Zeiss | 426559-0000-000 | |
Other | EC PlnN 10x objective | Zeiss | 420341-9911-000 | |
Other | Motorized tissue homogenizer | RPI | 299200 | |
Other | Fisher Scientific Sonic Dismembrator | Fisher Scientific | Model 500 | |
Other | Refrigerated Centrifuge | Eppendorf | 5424R | |
Other | Photospectrometer | Molecular Devices | SpectraMax M5e | |
Other | PVDF membrane | EMD Millipore | IPVH00010 | |
Other | LI-COR C-Digit | Li-Cor Biosciences | CDG-001313 | |
Other | 70 µm cell strainer | Corning | 352350 | |
Other | Ammonium-Chloride- Potassium (ACK) Lysing Buffer | Gibco | A1049201 | |
Other | Clinical Centrifuge | Beckman Coulter | Allegra 6R | |
Other | 40 µm filter | BD Falcon | 352340 | |
Other | Myelin Removal Beads | Miltenyi | 130-096-731 | |
Other | MidiMACS separator | Miltenyi | 130-042-302 | |
Other | LS Columns | Miltenyi | 130-042-401 | |
Other | Hemacytometer | Millipore Sigma | Z359629 | |
Other | Chromium Next GEM Chip G | 10X Genomics, Inc | NC1000127 | |
Other | 10X Genomic Chromium Controller | 10X Genomics, Inc | N/A | |
Other | NovaSeq Illumina 6000 | Illumina | N/A | |
Other | Cryostat | Leica Biosystems | CM3050S | |
Other | 70 µm Cell Strainer | Corning | 352350 | |
Other | Confocal Microscope | Nikon | C1 | |
Other | Confocal Microscope | Leica Biosystems | SP8 | |
Genetic Reagent (Mus musculus C57BL/6) | Csf2-/- | Jackson Laboratories | Stock No: 026812 | PMID:8171324 MGI: J:17978 |
Genetic Reagent (Mus musculus C57BL/6) | ROSA26-TdTom | Jackson Laboratories | Stock No: 007576 | PMID:17868096 MGI: J:124702 |
Genetic Reagent (Mus musculus C57BL/6) | CD45.1 | Jackson Laboratories | Stock No: 002014 | PMID:11698303 MGI: J:109863 PMID:11994430 MGI: J:109854 PMID:12004082 MGI: J:109853 |
Genetic Reagent (Mus musculus C57BL/6) | Wildtype, WT | Taconics | B6NTac | |
Genetic Reagent (Mus musculus C57BL/6) | Arg1-eYFP | Jackson Laboratories | Stock No: 015857 | PMID:17450126 MGI: J:122735 |
Oligonucleotide | Csf2 Forward | Integrated DNA Technologies | N/A | 5’-GTGAAACACAAGTTACCACCTATG-3’ |
Oligonucleotide | Csf2 Reverse | Integrated DNA Technologies | N/A | 5’-TTTGTCTTCCGCTGTCCAA-3’ |
Oligonucleotide | Neomycin Forward | Integrated DNA Technologies | N/A | 5’-CTTGGGTGGAGAGGCTATTC-3’ |
Oligonucleotide | Neomycin Reverse | Integrated DNA Technologies | N/A | 5’-AGGTGAGATGACAGGAGATC-3’ |
Software | WIS-Neuromath | Weizmann Institute of Science | Version 3.4.8 | PMID:23055261 |
Software | Image Studio Software | Li-Cor Biosciences | Version 5.2.5 | |
Software | NovaSeq control software | Illumina | Version 1.6 | |
Software | Real Time Analysis (RTA) software | Illumina | Version 3.4.4 | |
Software | Cellranger | 10X Genomics, Inc | Version 3.1.0 | |
Software | FACSDiva | BD Biosciences | Version 7 | |
Software | FlowJo | FlowJo LLC | Version 10.6.2 | |
Software | Seurat | Satija Lab - New York Genome Center | Version 3.1.2 | |
Software | R | r-project.org | Version 3.6.2 | |
Software | Slingshot | Bioconductor | Version 1.4.0 | |
Software | Ranger | Comprehensive R Archive Network | Version 0.12.1 | |
Software | Prism | GraphPad | Version 7 and 8 | |
Software | Imaris | Bitplane | ||
Software | Leica Application Suite (LAS X) | Leica | ||
Software | Zen Application Software | Zeiss | Pro 3.8 | |
Other | SomnoSuite | Kent Scientific | SS-01 | |
Other | Povidone-Iodine Prep Pad | PDI Healthcare | B40600 | |
Other | Alcohol Prep, Sterile, Md, 2 Ply | Covidien | 6818 | |
Other | Fine Forceps Dumont #55 Dumoxel | Roboz Surgical Instrument | RS-5063 | |
Other | 7 mm Reflex Wound Clips | Cell Point Scientific | 203–1000 | |
Other | Micro Friedman Rongeur | Roboz Surgical Instrument | RS-8306 | |
Other | McPherson-Vannas Micro Dissecting Spring scissors | Roboz Surgical Instrument | RS-5600 | |
Other | COATED VICRYL (polyglactin 910) Suture | Ethicon | J463G | |
Other | dumont #7 curved forceps | Fine Science Tools | 11271–30 | |
Other | Miltex halsted mosquito forceps | Integra LifeSciences | 724 | |
Other | Nanofil 10 µL syringe | World Precision Instruments | NANOFIL | |
Other | 36 g beveled nanofil needle | World Precision Instruments | NF36BV-2 | |
Other | Non-absorbable sutures | Ethicon | 640G | |
Other | Absorbable sutures | Ethicon | J463G |