Journal: The Journal of Experimental Medicine

Article Title: Mast cells acquire MHCII from dendritic cells during skin inflammation

doi: 10.1084/jem.20160783

Figure Lengend Snippet: MCs incorporate donor DC-derived H2 b MHCII protein complexes. (A) SJL/J B6 BM chimera were generated by BM transplantation from C57BL/6 donor mice (CD45.2 + H2 b MHCII) to SJL/J recipients (CD45.1 + H2 s MHCII). (B) CD45.2 + expression on skin DCs from SJL/J mice ( n = 6) and SJL/J B6 BM chimera 24 h after DNFB ( n = 8) was assessed by flow cytometry. (C) CD45.1 + expression on skin MCs from SJL/J mice ( n = 6) and SJL/J B6 BM chimera 24 h after DNFB ( n = 8). (D–F) Surface CD45.2 and intracellular H2 b MHCII expression by CD45.1 + recipient ear skin MCs (c-kit + FcεRI + ) of SJL/J mice (D) and SJL/J B6 BM chimera (E) 24 h after DNFB was measured by flow cytometry and quantified as box plot (F). ***, P < 0.001.

Article Snippet: 6-wk-old SJL/J recipient mice (CD45.1 + MHCII haplotype H2 s ) were lethally irradiated (whole body irradiation, 9 Gy; x-ray source, MaxiShot, Yxlon) before retro-orbital i.v. injection of 20 × 10 6 whole BM cells isolated from age- and sex-matched C57BL/6 donor mice (CD45.2 + MHCII haplotype H2 b ).

Techniques: Derivative Assay, Generated, Transplantation Assay, Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Mast cells acquire MHCII from dendritic cells during skin inflammation

doi: 10.1084/jem.20160783

Figure Lengend Snippet: DC-instructed MCs efficiently induce ex vivo allogeneic T cell priming. (A) SJL/J B6 chimera were generated by lethal irradiation of SJL/J recipient mice and transfer of BM cells from C57/BL6 donor mice. DC-to-MC communication was induced by DNFB administration onto ear skin of SJL/JB6 chimera mice or control SJL/J mice, and DCs and MCs were sorted from ear skin 24 h after DNFB. CFSE-labeled SJL/J T cells were cocultured with DCs (CD45.2 + CD11c + H2b + ) and MCs (CD45.1 + c-kit + FcεRI + ) sorted from ear skin of SJL/J B6 chimera mice ( n = 9) or SJL/J control mice ( n = 6) 24 h after DNFB, or C57BL/6 spleen DCs ( n = 6) as positive control. (B and C) T cell proliferation was assessed as CFSE dilution (B) and quantified as proliferated T cell fraction from total T cells and as mean fluorescence intensity (MFI; C). (D) The proliferated T cell fraction was plotted versus the H2 b -expressing MC fraction to assess parameter correlation. (E) Cytokines released upon SJL/J T cell co-culture with DCs and MCs were quantified in the co-culture supernatant by bead-based multiplex assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: 6-wk-old SJL/J recipient mice (CD45.1 + MHCII haplotype H2 s ) were lethally irradiated (whole body irradiation, 9 Gy; x-ray source, MaxiShot, Yxlon) before retro-orbital i.v. injection of 20 × 10 6 whole BM cells isolated from age- and sex-matched C57BL/6 donor mice (CD45.2 + MHCII haplotype H2 b ).

Techniques: Ex Vivo, Generated, Irradiation, Labeling, Positive Control, Fluorescence, Expressing, Co-Culture Assay, Multiplex Assay

Journal: Nature

Article Title: Circulating myeloid-derived MMP8 in stress susceptibility and depression

doi: 10.1038/s41586-023-07015-2

Figure Lengend Snippet: a , Gating strategy for live CD45 + cells for the Cytometry by time of flight (CyTOF) experiment ( panels a - i ). b , Social interaction ratio ( n = 10 CON, 10 SUS and 9 RES) and c , locomotion ( n = 10 CON, 10 SUS and 9 RES) (behavioural data from blood CyTOF experiment). d , Marker expression and assigned leukocyte subpopulations in blood. e , Frequencies of Ly6C low monocytes ( n = 10 CON] 9 SUS and 8 [RES]), dendritic cells ( n = 9 CON, 9 SUS and 9 [RES]), monocyte-derived dendritic cells ( n = 9 CON, 10 SUS and 9 RES), natural killer cells ( n = 9 CON, 9 SUS and 8 RES), CD4 + T cells ( n = 10 CON, 9 SUS and 9 RES), CD8 + T cells (CD44 hi , CD62L low ) ( n = 10 CON, 10 SUS and 9 RES), CD8 + T cells (CD44 hi , CD62L hi ) cells ( n = 10 CON, 9 SUS and 9 RES), CD8 + T cells (CD44 low , CD62L hi ) ( n = 10 CON, 10 SUS and 9 RES), CD8 + T cells (CD44 low , CD62L low ( n = 10 CON, 10 SUS and 9 RES), eosinophils ( n = 10 CON, 10 SUS and 8 RES) and undefined ( n = 9 CON, 10 SUS and 9 RES). f , Social interaction ratio ( n = 6 CON, 8 SUS and 7 RES) and g , locomotion ( n = 6 CON, 8 SUS and 7 RES) (behavioural data from brain CyTOF experiment). h , Marker expression and assigned leukocyte subpopulations of CD45 + brain leukocytes. i , Frequencies of microglia ( n = 6 CON, 8 SUS and 7 RES), border-associated macrophages ( n = 6 CON, 8 SUS and 7 RES), dendritic cells ( n = 6 CON, 8 SUS and 7 RES), natural killer cells ( n = 5 CON, 8 SUS and 7 RES), B cells ( n = 6 CON, 8 SUS and 7 RES), Ly6C low monocytes ( n = 6 CON, 8 SUS, & 7 RES), neutrophils ( n = 6 CON, 7 SUS, & 7 RES), CD4 + T cells ( n = 6 CON, 8 SUS, & 7 RES), CD8 + T cells ( n = 6 CON, 8 SUS and 7 RES) in whole brain tissue homogenates. Flow cytometry assessment of j , Ly6C hi monocytes ( n = 5 CON, 7 SUS and 5 RES), neutrophils ( n = 5 CON, 7 SUS and 5 RES), T cells ( n = 5 CON, 7 SUS and 5 RES), B cells ( n = 5 CON, 7 SUS and 5 RES) in leptomeninges, k , Ly6C hi monocytes ( n = 5 CON, 7 SUS and 5 RES), neutrophils ( n = 5 CON, 7 SUS and 5 RES), T cells ( n = 5 CON, 7 SUS and 5 RES), B cells in dura ( n = 5 CON, 7 SUS and 5 RES), l , Ly6C hi monocytes ( n = 5 CON, 7 SUS and 5 RES), neutrophils ( n = 5 CON, 6 SUS and 5 RES), T cells ( n = 5 CON, 7 SUS and 5 RES), B cells ( n = 5 CON, 7 SUS and 5 RES) in choroid plexus. (One-way ANOVA with Bonferroni post hoc test panels b , c , e , f , g , i - l ; for blood, each data point represents one biological sample; for brain, each data point represents four pooled brains; for leptomeninges, dura and choroid plexus, each data point represents three pooled samples). * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± s.e.m. Abbreviations: moDCs: Monocyte-derived dendritic cells; NK cells: Natural killer cells; BAM: Border-associated macrophages. Detailed statistics are in Supplementary Table .

Article Snippet: The level of chimerism was assessed using flow cytometry, comparing CD45.1 (host) (mouse anti-CD45.1-PE-Cyanine7, clone A20, Invitrogen, 25-0453-81) and CD45.2 (donor) (mouse anti-CD45.2-BV421, clone 104, BD Bioscience, 562895) leukocytes, and measuring MMP8 in plasma (Abcam, ab206982).

Techniques: Cytometry, Marker, Expressing, Derivative Assay, Flow Cytometry

Journal: Nature

Article Title: Circulating myeloid-derived MMP8 in stress susceptibility and depression

doi: 10.1038/s41586-023-07015-2

Figure Lengend Snippet: a , Experimental outline of CyTOF experiment. SI, social interaction. b , h , t -Distributed stochastic neighbour embedding ( t -SNE) maps of CD45 + cells in blood ( b ) and brain ( h ). The colour of each cluster corresponds to the assigned cell type. BAMs, border-associated macrophages; moDCs, monocyte-derived dendritic cells; NK, natural killer. c , Frequencies of Ly6C hi monocytes ( n = 10 CON, 12 SUS and 10 RES), neutrophils ( n = 10 CON, 10 SUS and 9 RES), naive B cells ( n = 10 CON, 10 SUS and 9 RES), transitional B cells and memory B cells ( n = 10 CON, 10 SUS and 9 RES) in circulation. One-way ANOVA with Bonferroni post hoc test. Each data point represents one biological sample; data are mean ± s.e.m. d , f , Number of monocytes ( d ) and neutrophils ( f ) in the circulation of patients with MDD compared with healthy controls (HC). n = 52 heathy controls and 131 MDD; two-tailed Student’s t -test. e , g , Correlation between perceived stress and monocyte numbers ( e ; n = 169 (HC and MDD)) and neutrophil numbers ( g ; n = 169 (HC and MDD)). Two-tailed Pearson correlation coefficient. i , Ly6C hi monocytes in whole brain. n = 6 CON, 8 SUS and 7 RES. One-way ANOVA with Bonferroni post hoc test; each data point represents four pooled brains. Data are mean ± s.e.m. j , Experimental outline of cell type-specific RNA sequencing of Ly6C hi and Ly6C low monocytes, B cells and T cells from blood. k , Number of differentially expressed genes (adjusted P value < 0.05 and log fold change > |1|) in Ly6C hi monocytes. l , The 25 most significantly differently expressed protein-coding genes in Ly6C hi monocytes from SUS versus CON mice. m , Top three gene ontology (GO) terms from significantly upregulated genes in Ly6C hi monocytes of SUS versus CON mice. k – m , P values adjusted for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. Detailed statistics are in Supplementary Table .

Article Snippet: The level of chimerism was assessed using flow cytometry, comparing CD45.1 (host) (mouse anti-CD45.1-PE-Cyanine7, clone A20, Invitrogen, 25-0453-81) and CD45.2 (donor) (mouse anti-CD45.2-BV421, clone 104, BD Bioscience, 562895) leukocytes, and measuring MMP8 in plasma (Abcam, ab206982).

Techniques: Derivative Assay, Two Tailed Test, RNA Sequencing Assay

Journal: Nature

Article Title: Circulating myeloid-derived MMP8 in stress susceptibility and depression

doi: 10.1038/s41586-023-07015-2

Figure Lengend Snippet: a , Chimerism (CD45.2 + vs. total CD45 + cells) of unstressed and stressed WT and Mmp8 −/− transplants ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 15 WT → WT-CSDS and 17 Mmp8 −/− → WT-CSDS). Frequencies of circulating b , Ly6C hi monocytes ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 15 WT → WT-CSDS and 17 Mmp8 −/− → WT-CSDS) and c , neutrophils ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 15 WT → WT-CSDS and 17 Mmp8 −/− → WT-CSDS). Plasma levels of d , Interleukin 6 (IL6) ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 12 WT → WT-CSDS and 12 Mmp8 −/− → WT-CSDS), e , IL10 ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 13 WT → WT-CSDS and 12 Mmp8 −/− → WT-CSDS), f , IL12p40 ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 13 WT → WT-CSDS and 13 Mmp8 −/− → WT-CSDS), g , C-C motif chemokine ligand 2 (CCL2) ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 13 WT → WT-CSDS and 13 Mmp8 −/− → WT-CSDS), h , Vascular endothelial growth factor (VEGF) ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 13 WT → WT-CSDS and 13 Mmp8 −/− → WT-CSDS) and i , Tumor necrosis factor (TNF) ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 12 WT → WT-CSDS and 13 Mmp8 −/− → WT-CSDS). j , Sucrose preference test ( n = 6 WT → WT-CON, 7 Mmp8 −/− →WT-CON, 14 WT → WT-CSDS and 15 Mmp8 −/− → WT-CSDS), k , splash test ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 14 WT → WT-CSDS and 16 Mmp8 −/− → WT-CSDS), and l , elevated plus maze test. Sickness associated behaviours such as m , body weight ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 15 WT → WT-CSDS and 17 Mmp8 −/− → WT-CSDS), n , food consumption ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 15 WT → WT-CSDS and 18 Mmp8 −/− → WT-CSDS) or o , locomotion ( n = 6 WT → WT-CON, 7 Mmp8 −/− → WT-CON, 15 WT → WT-CSDS and 17 Mmp8 −/− → WT-CSDS). (Two-way ANOVA followed by Tukey’s post hoc testing [ for all panels ]). Data are shown as mean ± s.e.m. Detailed statistics are in Supplementary Table .

Article Snippet: The level of chimerism was assessed using flow cytometry, comparing CD45.1 (host) (mouse anti-CD45.1-PE-Cyanine7, clone A20, Invitrogen, 25-0453-81) and CD45.2 (donor) (mouse anti-CD45.2-BV421, clone 104, BD Bioscience, 562895) leukocytes, and measuring MMP8 in plasma (Abcam, ab206982).

Techniques:

Journal: Cell reports

Article Title: m 6 A RNA Methylation Maintains Hematopoietic Stem Cell Identity and Symmetric Commitment

doi: 10.1016/j.celrep.2019.07.032

Figure Lengend Snippet: (A) Mettl3 cKO HSCs are less quiescent. Representative flow cytometry plots for assessing cell cycle status of Mettl3 f/f and Mettl3 cKO HSC by Pyronin Y staining. (B) Quantification of cell cycle analysis. n = 5. (C) Increased mitochondrial mass in Mettl3 cKO HSCs. Representative histograms of Mitotracker Green staining in Mettl3 f/f and Mettl3 cKO HSCs. (D) Mitochondrial mass of different HSPC population was evaluated by Mitotracker Green staining quantified by flow cytometry. n = 5. (E) Scheme of transplant strategy. Non-competitive reconstitution assay in which 10 6 donor BM cells from Mettl3 f/f or Mettl3 cKO mice at 3 weeks post-plpC were transplanted into CD45.1 congenic recipient mice. (F) Mettl3 cKO bone marrow fails to reconstitute hematopoietic compartments in recipient mice. CD45.2 chimerisms of HSCs, MPPs and progenitor compartments from (E) were analyzed by flow cytometry at 16 weeks post-transplant. n = 10. (G) Scheme of the experimental procedure in (H) and (I). CD45.1 recipient mice were transplanted with Mettl3 flox/flox Cre − or Cre + bone marrow cells (pre-plpC). At 6 weeks post-transplantation, CD45.1 congenic recipient mice were then injected with plpC to deplete METTL3. (H) The reconstitution defect in Mettl3 cKO bone marrow is cell-autonomous. CD45.2 chimerism analysis of HSCs, MPPs, and progenitor compartments from experiment (G) at 3 weeks post-pIpC. n = 5. (I) Frequencies of HSCs and MPPs in the donor CD45.2 population from experiment (G). n = 5. Mean and SEM are shown (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). n represents number of mice.

Article Snippet: For transplanted mice, we added CD45.1-PE-Txase Red (BD Bioscience, 562452) and CD45.2-A700 (Invitrogen, 56–0454-82) to distinguish donor and recipient cells.

Techniques: Flow Cytometry, Staining, Cell Cycle Assay, Reconstitution Assay, Transplantation Assay, Injection

Journal: Cell reports

Article Title: m 6 A RNA Methylation Maintains Hematopoietic Stem Cell Identity and Symmetric Commitment

doi: 10.1016/j.celrep.2019.07.032

Figure Lengend Snippet: (A) Experimental scheme for acute knockdown of Mettl3 in LSK cells by siRNA followed by transplantation. (B) qRT-PCR of Mettl3 to confirm the knockdown efficiency in LSK cells from (A). (C) Donor engraftments ofCD45.2 in different lineage compartments were analyzed by flow cytometry in peripheral blood at 4 weeks post-transplant from (A). ctrl n = 10; siMettl3 n = 5. (D) CD45.2 chimerism analysis of HSCs, MPPs, and progenitor compartments in bone marrow from experiment (A) at 12 weeks post-transplant. ctrl n = 10; siMettl3 n = 5. (E) CD45.2 chimerism analysis of different lineage populations in bone marrow from experiment (A) at 12weeks post-transplant. ctrl n = 10; siMettl3 n = 5. Mean and SEM are shown (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). n represents number of mice.

Article Snippet: For transplanted mice, we added CD45.1-PE-Txase Red (BD Bioscience, 562452) and CD45.2-A700 (Invitrogen, 56–0454-82) to distinguish donor and recipient cells.

Techniques: Transplantation Assay, Quantitative RT-PCR, Flow Cytometry

Journal: Cell reports

Article Title: m 6 A RNA Methylation Maintains Hematopoietic Stem Cell Identity and Symmetric Commitment

doi: 10.1016/j.celrep.2019.07.032

Figure Lengend Snippet: (A) Significant differentially expressed genes (padj < 0.05) in cKO HSCs compared to Mettl3 f/f were shown as heatmap. (B) Differentially expressed genes in Mettl3 cKO HSCs were enriched with Mettl3 KO ESC and RBM15 KO LSK expression signatures. Enrichr analysis using upregulated genes in METTL3-depleted HSCs from bulk RNA-seq. (C) Unbiased gene set enrichment analysis using 4,733 curated gene sets against the rank list of differential expressed genes between Mettl3 f/f and cKO HSCs. Self-renewal signature was negatively enriched in Mettl3 cKO HSCs. Translation and ribosome gene sets were positively enriched in Mettl3 cKO HSCs. (D) Increased global translation in Mettl3 cKO HSCs. Representative histograms (top) and quantification (bottom) of OP-Puro incorporation into sorted Mettl3 f/f and cKO HSCs. (E) Scheme of transplant strategy in (F). Sorted HSCs and MPP1s from Mettl3 f/f and Mettl3 cKO mice were injected into CD45.1 recipient mice with CD45.1 competitor BM cells. (F) Mettl3 cKO HSCs function as MPP1s in competitive transplant assays. Engraftment of CD45.2 donor cells was analyzed in HSPCs from recipient mice at 6 weeks. n = 10. (G) Engraftment of CD45.2 donor cells was analyzed in HSPCs from recipient mice at 40 weeks. n = 10. (H) Spearman correlation among HSC and MPP1 populations from Mettl3 f/f and Mettl3 cKO mice. (I) Gene set enrichment analysis of up-or downregulated genes in Mettl3 cKO HSC identified by bulk RNA-seq, against the rank list of differentially expressed genes between Mettl3f/f HSC and Mettl3f/f MPP1. Mean and SEM are shown (*p<0.05, **p<0.01, ***p<0.001, and ****p < 0.0001). n represents number of mice.

Article Snippet: For transplanted mice, we added CD45.1-PE-Txase Red (BD Bioscience, 562452) and CD45.2-A700 (Invitrogen, 56–0454-82) to distinguish donor and recipient cells.

Techniques: Expressing, RNA Sequencing Assay, Injection

Journal: Cell reports

Article Title: m 6 A RNA Methylation Maintains Hematopoietic Stem Cell Identity and Symmetric Commitment

doi: 10.1016/j.celrep.2019.07.032

Figure Lengend Snippet: (A) Scheme of experiment strategy. LSK cells were sorted from Mettl3 f/f and Mettl3 cKO mice and transduced with control and MYC overexpression retrovirus. Donor cells were then transplanted into CD45.1 recipient mice with CD45.1 competitor BM cells. (B) Quantification of the frequency of donor-derived cells was shown in LSK and MP populations, n = 9. n represents number of mice. (C) MYC overexpression rescues the repopulating defect of Mettl3 cKO LSKs. Representative flow cytometry plots of engraftment of donor-derived CD45.2 cells. (D) METTL3 overexpression rescue MYC expression defect in Mettl3 cKO HSCs as quantified by immunofluorescence. (E and F) Paired daughter cell assay using sorted HSCs from Mettl3 f/f and Mettl3 cKO mice transfected with vectors expressing METTL3 or METTL3-CD or empty vector as control to indicate MYC (E) and NUMB (F) expression. Pie chart shows different types of cell divisions in HSCs as indicated. Number of daughter pairs assessed: Mettl3 f/f +Vec, n = 141; cKO +Vec, n = 35; cKO +METTL3 WT, n = 45; and cKO +METTL3 CD, n = 65.n represents number of paired HSCs measured. Mean and SEM are shown (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Article Snippet: For transplanted mice, we added CD45.1-PE-Txase Red (BD Bioscience, 562452) and CD45.2-A700 (Invitrogen, 56–0454-82) to distinguish donor and recipient cells.

Techniques: Transduction, Over Expression, Derivative Assay, Flow Cytometry, Expressing, Immunofluorescence, Transfection, Plasmid Preparation

Journal: Cell reports

Article Title: m 6 A RNA Methylation Maintains Hematopoietic Stem Cell Identity and Symmetric Commitment

doi: 10.1016/j.celrep.2019.07.032

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For transplanted mice, we added CD45.1-PE-Txase Red (BD Bioscience, 562452) and CD45.2-A700 (Invitrogen, 56–0454-82) to distinguish donor and recipient cells.

Techniques: Recombinant, SYBR Green Assay, Flow Cytometry, Multiplex Assay, Expressing, Software

Journal: Cell reports

Article Title: Epithelial-myeloid exchange of MHC class II constrains immunity and microbiota composition

doi: 10.1016/j.celrep.2021.109916

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: OT-II transgenic mice (stock # 004194) and CD45.1 mice (stock # 002014) were purchase from Jackson Laboratories and crossed to create OT-II;CD45.1 donor-mice which were confirmed by genotyping according to Jackson lab’s protocol for the OT-II transgenes and blood phenotyping with anti-CD45.1 and anti-CD45.2 antibodies (Tonbo Biosciences, clone A20 and clone 104).

Techniques: Virus, Recombinant, SYBR Green Assay, Cell Isolation, DNA Extraction, Enzyme-linked Immunosorbent Assay, Sequencing, Expressing, Software, Luminex