Journal: Cell

Article Title: Functional characterization of the dural sinuses as a neuroimmune interface.

doi: 10.1016/j.cell.2020.12.040

Figure Lengend Snippet: Figure 4. Dural sinus-associated stroma allows homeostatic T cell recruitment and retention (A) t-SNE visualization of color-coded scRNA-seq of whole dural meningeal populations based on cell types, n = 10 total pooled mice per sample, from two independent experiments. (B) t-SNE visualization of color-coded scRNA-seq analysis of whole dural meningeal populations based on predicted adhesiveness to mural or endothelial populations using RNAMagnet. Colored cells represent the strongest predicated physical adhesion of that cell to an endothelial (cyan) or mural (magenta) cell. Gray cells represented maximal predicted adhesiveness to non-stromal populations. Opacity represents strength of adhesion. (C) t-SNE visualization of expression for selected ligand (cyan) and receptor (magenta) pairings for stromal-immune physical interactions and signaling crosstalk. (D–F) Immunohistochemistry and quantification of collagen I and laminin coverage in non-sinus and sinus regions of young (2–3 months old) and old (20– 24 months old) dural meninges. NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA with Sidak’s post hoc test), n = 3 mice/group. (G) Dot plot demonstrating scaled gene expression and percentage of cells expressing these genes for chemokines in meningeal stromal populations from scRNA-seq stromal analysis. (H) Immunohistochemistry of CXCL12 expression at sinus and non-sinus regions in WT mice, representative of n = 4 mice. (IandJ)ImmunohistochemistryandquantificationofCXCL12expressionatsinusandnon-sinusregionsinCXC12-DsRedmice.***p<0.001(Student’sttest),n=4mice. (K) Ratio of non-targeting control (NTC) or CXCR4 KO CRISPR-Cas9 modified CD4 T cells 12 days after transfer into RAG2 KO mice at a 1:1 ratio in the lung, spleen, and dural meninges. Numbers are normalized to the ratio of NTC and CXCR4 KO cells in the blood of the same mouse. *p < 0.05, **p < 0.01 (one-way ANOVA with Tukey’s post hoc test), n = 6. (L–N) Flow cytometry analysis and quantification of viable, single, CD45+ Thy1.2+ TCRb+ CD4+ T cells in the dura and blood of PDGFRb-CreERT2::CXCL12+/+ or PDGFRb-CreERT2::CXCL12fl/+ mice 60 days after final tamoxifen injection (five injections). NS, not significant, **p < 0.01 (Student’s t test), n = 3–5 per group. See also Figures S5 and S6 and Table S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Zombie NIR Fixable Viability Kit BioLegend Cat#423106 Zombie Aqua Fixable Viability Kit BioLegend Cat#423102 LIVE/DEAD Fixable Blue Dead Cell Stain Kit Thermo Fisher Ca#L23105 Purified anti-Mouse CD16/CD32 (Fc block) BioLegend Cat#101302 Seurm-FBS Certified US origin Thermo Fisher Cat#16000044 eBioscience Cell Stimulation Cocktail eBioscience Cat#00-4970-03 Brefeldin A Solution eBioscience Cat#00-4506-51 Foxp3 / Transcription Factor Staining Buffer Set Kit eBioscience Cat#00-5523-00 LS columns Miltenyi Biotec Cat#130-042-401 MS columns Miltenyi Biotec Cat#130-042-201 Purified NA/LE Hamster Anti-Mouse CD28 BD Biosciences Cat#553294 Purified NA/LE Hamster Anti-Mouse CD3e BD Biosciences Cat#553057 Alt-R CRISPR-Cas9 Negative Control crRNA #1 IDT Cat#1072544 Alt-R CRISPR-Cas9 tracrRNA IDT Cat#1072532 TrueCut Cas9 Protein v2 Thermo Fisher Cat#A36499 Recombinant Murine IL-2 PeproTech Cat#212-12-500ug Corning HTS Transwell 96 well permeable supports Corning Cat#CLS3388 Recombinant Murine SDF-1a (CXCL12) PeproTech Cat#250-20A InVivoMAb rat IgG1 isotype control BioXCell Cat#BE0088 InVivoMAb rat IgG2b isotope control BioXCell Cat#BE0090 InVivoMAb anti-mouse/human VLA4 BioXCell Cat#BE0071 InVivo mAb anti-mouse LFA1a BioXCell Cat#BE0005-1 InVivoMAb anti-mouse PSGL-1 BioXCell Cat#BE0186-5MG Maxpar Fix and Perm Buffer Fluidigm Cat#201067 Maxpar Cell Staining Buffer Fluidigm Cat#201068 Cell-ID Cisplatin Fluidigm Cat#201064 Maxpar Nuclear Antigen Staining Buffer Set Fluidigm Cat#201063 Cell-ID Intercalator-Ir—125 mM Fluidigm Cat#201192A Invitrogen Ambion UltraPure BSA Thermo Fisher Cat#AM2616 Trypan blue, 0.4% Thermo Fisher Cat#15250061 Microcon-30kDa Centrifugal Filter Unit with Ultracel-30 membrane Millipore Cat#MRCF0R030 Critical commercial assays CD4+ T Cell Isolation Kit, mouse Miltenyi Biotec Cat#130-104-454 CD11c MiroBeads, UltraPure, mouse Miltenyi Biotec Cat#130-125-835 Cell-ID 20-Plex Pd Barcoding Kit Fluidigm Cat#201060 Chromium Single Cell 30 Library & Gel Bead Kit v2 10X Genomics Cat#PN-120267 Chromium Single Cell 30 Library & Gel Bead Kit v3 10X Genomics Cat#PN-120267 Amaxa P4 Primary Cell 4D-Nucleofector Kit Lonza Cat#V4XP-4024 Pierce Quantitative Fluorometric Peptide Assay Thermo Fisher Cat#23290 RNAscope Multiplex Fluorescent Reagent Kit V2 Advanced Cell Diagnostics Cat#323110 Tyramide Signal Amplification (TSA) Plus Fluorescence Palette Kit Perkin Elmer Cat#NEL760001KT (Continued on next page) ll Cell 184, 1000–1016.e1–e16, February 18, 2021 e5 Article

Techniques: Expressing, Immunohistochemistry, Gene Expression, Control, CRISPR, Flow Cytometry, Injection