Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 1 Characterization of human bulge cell markers. (A) CD200+ cells were stained in red by immunohistochemistry (magnification = 100·). Black rectangle identifies boundaries of 400· magnification shown in left inset. Positive cells are located in the outer root sheath of the hair follicle (HF), between insertions of sebaceous glands (SG) and arrector pili muscle (AP). Asterisk shows lumen of the HF (hair shaft was lost during staining). (B) Consecutive sections of human scalp skin biopsies from young individuals were stained for KRT15, CD200, and KRT19 by immunohistochemistry. Upper and lower limits of CD200 positive staining superimpose with those of KRT19 and KRT15 respectively (highlighted by black lines). Daggers indicate hair shafts remnants. (C) Schematic representation of localization of protein used as human bulge cell markers. Bulge cells are CD200+ ⁄ KRT15+ ⁄ KRT19+.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Staining, Immunohistochemistry

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 2 Characterization of isolated human bulge cells from young human skin. (A) CD200, KRT15 and KRT19 mRNA levels were measured by Q-PCR on CD200+ (bulge) and CD200) (differentiated) cells of the hair follicle (HF) in young (< 40 years) individuals. N = 6; *P < 0.05. (B) Transglutaminase 1 (TGM1) mRNA levels in same cell populations. N = 3; P = 0.022.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Isolation

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 3 Additional markers of young human bulge keratinocytes. (A, B) Frozen scalp skin sections from young individuals were immunostained for CD34 (A) and follistatin (B). Original magnification = 100·. Black rectangle indicates location of left inset (magnification 400·). Black arrows represent upper and lower limits of bulge area. Representative of at least four individuals. (C) Quantitative expression of gene transcripts for various potential hair follicle (HF) stem cell markers in bulge (CD200+) and differentiated (CD200)) cells of the HF in young human skin. N = 6; *P < 0.05; NS = not significant vs. bulge cells. Gas1, EGFR, CCN2 and S100A4 are represented in separate bar graphs to account for higher basal expression levels.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Expressing

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 5 Aging does not alter bulge marker expression in human skin. Total RNA from bulge (CD200+) and differentiated (CD200)) cells of the hair follicle (HF) were isolated from scalp skin biopsies of young (< 40 years) and aged (> 70 years) individuals, and analyzed by Q-PCR for human HF stem cell markers. (A) Quantitative expression of CD200, KRT15 and KRT19 human bulge cell markers. (B) Direct comparison of gene transcript levels of various potential additional bulge cell markers between young and aged individuals. Gas1, EGFR and CCN2 are represented in separate bar graphs to account for higher expression levels. N = 6; *P<0.05; NS = not significant vs. young counterpart.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Marker, Expressing, Isolation, Comparison

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 6 Hedgehog (Hh) pathway components are selectively expressed in human bulge cells vs. differentiated hair follicle (HF) keratinocytes (KCs). Transcript levels of Hh pathway components and target genes were quantified by Q-PCR in bulge (CD200+) and differentiated (CD200)) cells of human HFs. N = 6; (A) young and (B) aged individuals. *P < 0.05; NS = not significant vs. differentiated HF KCs. (C) Quantitative expression of levels of Hh pathway components and target genes transcripts in bulge cells of young and aged individuals. NS = not significant vs. young counterpart.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Expressing

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 7 The hedgehog transmembrane receptor and target gene product Ptch is expressed in CD200+ cells of the bulge area of human hair follicles. (A, B) Ptch protein expression was stained by immunohistochemistry 100· magnification (A). Black rectangle in A identifies boundaries of 200· magnification shown in B. (C) Double immunofluorescence staining of human bulge area. Human scalp sections were stained with CD200 (green) and Ptch (red). Overlay shows that Ptch is expressed by CD200+ cells of the human bulge area. Magnification = 400·. Representative of at least three individuals.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Mass spectrometry analysis of CD11b immunoprecipitates from BMDMs. Left: experimental workflow for isolating the CD11b-interacting proteins. Right: relative abundance of CD11b-interacting proteins, with a focus on cell surface receptors with inhibitory potential, indicated by a color gradient. ITIM, immunoreceptor tyrosine-based inhibitory motif; ITSM, immunoreceptor tyrosine-based switch motif; NPXY, asparagine, proline, any residue, tyrosine. IP, immunoprecipitation. b Immunoblot analysis of CD11b and CD200R1 interaction in immunoprecipitates from WT, Itgb2 −/− and Cd200r1 −/− BMDMs. Relative abundance is shown at the bottom of each panel. c Flow cytometry analysis of CD200 expression on parental (top) and Tac expression on Tac + (bottom) mouse tumor cell lines. Red curves represent staining with CD200 or Tac mAbs. Filled curves, control (Ctrl) mAbs. d Microscopy-based phagocytosis assay of non-opsonized (−) or IgG-opsonized (+) Tac + WEHI-231, Tac + A20, Tac + J558 and Tac + TUBO cells by WT BMDMs, in presence of CD200 mAb OX-90 (rat IgG2a) or Ctrl mAb 2A3 (rat IgG2a). Tumor cells were opsonized with Tac mAb 7G7 (mouse IgG2a), ( n = 3). e Time-course pHrodo-based phagocytosis assay using IgG-opsonized Tac + WEHI-231 cells and WT BMDMs. WEHI-231 cells were labeled with pHrodo red dye, and BMDMs were labeled with CSFE. Phagocytosis over time (0-4 h) was analyzed using an IncuCyte Live Cell Analyzer. Left: representative images at 2 h (scale bar, 100 μm; arrows, BMDMs with engulfed tumor cells). Right: quantification of cumulative phagocytosis (top) and time-specific increase in phagocytosis (Δ phagocytosis; bottom) over 0-4 h, ( n = 3). f Confocal microscope-based conjugate formation and actin polarization assay of IgG-opsonized WEHI-231 cells labeled with CSFE (green) and co-incubated with WT BMDMs labeled with Cell Trace Violet (CTV; blue), in presence of CD200 mAb or Ctrl mAb. Actin (red) was detected by β-actin mAb. Left: representative images (scale bar, 10 μm; arrows, BMDMs with fully polarized actin). Scale, 10 μm. Right: quantification of conjugate formation (top) and of conjugates with fully polarized actin (bottom), ( n = 3). g As per Fig. 1d, except that phagocytosis of complement (C3bi)-opsonized WEHI-231 cells by WT BMDMs in the presence of blocking CD200 mAb OX-90, blocking CD11b mAb 5C6 (rat IgG2b), Ctrl mAb 2A3 or Ctrl mAb LTF-2 (rat IgG2b), ( n = 3). h As per Fig. 1d, expect that phagocytosis of IgG-opsonized WEHI-231 cells by WT BMDMs in the presence of CD200R1 mAb OX-131 (mouse IgG1, Fc-silent) or Ctrl mAb (mouse IgG1, Fc-silent), ( n = 3). Data are from three ( a – h ) independent experiments. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Statistical analysis: two-tailed t -test ( d – f , h ) with multiple comparisons ( e ); One-way ANOVA test with multiple comparisons ( g ); ns, not significant. See also Supplementary Fig. and Supplementary Table .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Mass Spectrometry, Residue, Immunoprecipitation, Western Blot, Flow Cytometry, Expressing, Staining, Control, Microscopy, Phagocytosis Assay, Labeling, Incubation, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200R1 expression (left) on WT (top) or Cd200r1 −/− (bottom) BMDMs, and CD200 expression (right) on Cd200 +/+ (top) or Cd200 −/− (bottom) WEHI-231 cells, as measured by flow cytometry. Red curves, CD200R1 or CD200 mAbs. Filled curves, Ctrl mAb. b As per Fig. , except that phagocytosis of IgG-opsonized WEHI-231 cells in the presence of WT or Cd200r1 −/− BMDMs was studied, ( n = 3). c As per Fig. 2b, except that phagocytosis of IgG-opsonized Cd200 +/+ and Cd200 −/− WEHI-231 cells, in the presence of WT or Cd200r1 −/− BMDMs, ( n = 3). Data are from three independent experiments ( a – c ). Each symbol represents one mouse. Data are mean ± s.e.m. Statistical analysis: two-way ANOVA test, with multiple comparisons ( b , c ), ns not significant. See also Supplementary Fig. .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a – g Tac + WEHI-231 tumor (with or without luciferase expression) were injected intravenously into Rag1 −/− mice, followed by intraperitoneal injection of Tac mAb combined with CD200 mAb or Ctrl mAb every 2 days. Mice were euthanized on day 15 ( b – d ) or monitored over time using luminescence, if cells were expressing luciferase ( e – g ), ( n = 12, b , c , n = 10, e – g ). a Schematic representation of the experimental workflow. I.V., intravenously; I.P., intraperitoneally. b Representative photographs of mice euthanized on day 15, with or without tumor injection, and treated with the indicated mAbs. Scale bar, 1 cm. c Liver weight of mice injected or not with tumor cells. d Hematoxylin and eosin staining of liver sections, showing blood vessels (blue asterisks) and tumor cell aggregates adjacent to blood vessels (outlined in white). Scale bars, 100 µm (10× magnification), and 20 µm (40× magnification). e Representative luminescence images on day 12. f Tumor progression over time as measured by luminescence. sec, second, st, steradian. g Kaplan–Meier analysis of survival. h – j Tac + A20 cells were injected subcutaneously in Rag1 −/− mice followed by intraperitoneal injection of Tac mAb combined with CD200 mAb or Ctrl mAb, every 2 days, ( n = 8). h Schematic representation of the experimental workflow. S.C, subcutaneously. i Tumor volume over time. j Tumor weight. Data are from three c or two d – j independent experiments, respectively. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Statistical analysis: one-way ANOVA test, with multiple comparisons ( c ); two-tailed t-test ( f , i , j ). log-rank (Mantel–Cox) test ( g ). ns, not significant. See also Supplementary Figs. and .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Luciferase, Expressing, Injection, IF-cells, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200R1 expression on human blood monocyte-derived macrophages, either unprimed or primed for 1 day with the indicated stimuli. Red curves, CD200R1 mAb. Filled curves, Ctrl mAb. Number in histograms indicated mean fluorescence intensity (MFI). b Fold changes of RNA expression of human blood monocyte-derived macrophages treated with or without IL-4. CD200R1 is highlighted in red. Fold changes (log 2 ; x -axis) and adjusted p -values (log 10 ; y-axis) are shown. The red line represents a 4-fold change threshold. Data are from dataset GSE195440 . c Frequency and relative expression levels of CD200R1 RNA across various human TAM subtypes, distinguished by gene expression profiles , . Data are from single-cell RNA sequencing (scRNA-seq) datasets GSE154763 and GSE146771 of TAMs from patients with the cancers indicated in Supplementary Fig. , and colon carcinoma. d RNA expression profiles of human hematological malignancies, determined by microarray analysis. Left: UMAP plots of samples with tumor diagnosis are shown and colored by cluster identity. Middle: CD200 and CD47 RNA expression levels overlaid onto the UMAP (color gradient shows relative expression). Right: mean expression of CD200 and CD47 RNA in common subtypes of hematological malignancies and normal immune cells (color gradient). Data are from published dataset HEMAP ( n = 7092). e Single-cell RNA sequencing analysis of human melanoma. Left: t-SNE profiles of melanoma malignant or non-malignant cells. Malignant melanoma cells are clustered by patient sample (top), while infiltrating immune cells are clustered by cell type (bottom). The middle and right: normalized expression of selected markers overlaid onto the t-SNE space, with a color gradient reflecting relative expression. Macrophages are outlined by a blue line. Data are from published dataset GSE115978 . f Same as d , except that expression of CD200 and CD47 RNA was analyzed in normal hematopoietic cells. g Flow cytometry analysis of expression of CD200 and CD47 on resting or activated human B cells, CD4 + T cells, and CD8 + T cells. Data are from three independent experiments ( a, g ). Each symbol represents one gene ( b ), one cell ( c ), one healthy donor or patient ( d ). Statistical analysis: Wald test followed by multiple comparisons ( b ); Kruskal–Wallis H test followed by Dunn’s post-hoc test with multiple comparisons ( c , left); two-tailed Mann–Whitney U test ( c , right panels). See also Supplementary Fig. .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Expressing, Derivative Assay, Fluorescence, RNA Expression, Gene Expression, RNA Sequencing, Microarray, Biomarker Discovery, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200 (top) or tumor antigens (CD20, CD38, CD123, DLL3 and SLAMF7; bottom) on various human tumor cell lines. Red curves, CD200 mAb or tumor antigen-specific mAbs. Filled curves, Ctrl mAbs. b As per Fig. , except using IL-4-primed human blood monocyte-derived macrophages and human tumor cells in the presence of samalizumab (human IgG1, Fc-silent) or Ctrl mAb MOPC21 (human IgG1, Fc-silent). IgG opsonization was performed using CD20 mAb rituximab (SLVL, 721.221), CD38 mAb daratumumab (NCI-H929), CD123 mAb talacotuzumab (KG-1a), DLL3 mAb rovalpituzumab (NCI-H209), or SLAMF7 mAb elotuzumab (SK-MEL-28) ( n = 3 or 4). c As per Fig. 5b, except using the indicated cell lines and samalizumab (human IgG1, Fc-silent), CD47 mAb B6H12 (human IgG1, Fc-silent), or Ctrl mAb MOPC21 (human IgG1, Fc-silent). Tumor cells were opsonized as detailed for Fig. 5b, ( n = 3). d –f Subcutaneous injection of SLVL cells in NSG mice, followed by intraperitoneal injection of rituximab (mouse IgG2a version) combined with samalizumab or Ctrl mAb every 2 days, ( n = 8). d Schematic representation of the experimental workflow. e Tumor volume over time. f Tumor weight. g –i Subcutaneous injection of 721.221 cells in NSG mice, followed by intraperitoneal injection of rituximab (mouse IgG2a version) combined with samalizumab or Ctrl mAbs every 2 days, ( n = 10). g Schematic representation of the experimental workflow. h Tumor volume over time. i Tumor weight. Data are from three to four ( a, b ), three ( c ), or two ( d-i ) independent experiments. Each symbol represents one human sample or one mouse. Data are presented as mean ± s.e.m. Statistical analysis: two-tailed t-test ( f , i ), with multiple comparisons ( b, c , e , h ). ns, not significant. See also Supplementary Fig. .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Derivative Assay, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Partial sequences of the cytoplasmic domain of CD200R1 from different species. The conserved tyrosines (Y 286 , Y 289 , and Y 297 ; based on mouse amino acid numbering) are in red, whereas the conserved NPxY motif is boxed. Identical residues are depicted by asterisks (*), while conserved and semi-conserved amino acids are highlighted by colons (:) and periods (.), respectively. b Phagocytosis of IgG-opsonized WEHI-231 cells by WT BMDMs expressing GFP alone, or Cd200r1 −/− BMDMs expressing GFP alone or CD200R1 variants, in the presence of CD200 mAb or Ctrl mAb, ( n = 3). c Mass spectrometry analyzes of cytoplasmic proteins with inhibitory potential interacting with synthetic biotinylated CD200R1 peptides, with or without phosphorylation at Y 286 or Y 297 in pull-down assays. Peptides are depicted at the top. Interactors identified by phosphorylated peptides are shown below. Negative regulators of immune cell activation, either adaptors, kinases, phosphatases or Ras-GAP, are indicated. d Immunoblot analysis of Dok-1 and Dok-2 expression (left) and phagocytosis of IgG-opsonized WEHI-231 cells (right) by BMDMs from WT or Dok1 −/− Dok2 −/− mice. β-actin as loading Ctrl (left). Normalized protein abundance (in %) relative to actin is shown below the top panel, ( n = 4). e Same as Fig. 6d except that WT BMDMs transduced with Ctrl or Csk-specific siRNAs [ Csk knockdown ( Csk KD )] were used. Two different Csk-specific siRNAs were studied. Csk expression (left) and phagocytosis (right) were studied, ( n = 4). f Summary of fold-changes in phagocytosis for the various genetically deficient BMDMs in response to CD200 mAb, compared to WT BMDMs, ( n = 3 or 4). g WT or Dok1 −/− Dok2 −/− BMDMs were stimulated or not for 1 min with biotinylated CD200R1 mAb OX-110 and streptavidin. Cell lysates were immunoprecipitated with α-Dok-1, α-Dok-2, α-Csk, or normal rabbit serum (NRS), and probed by immunoblotting with antibodies targeting phosphotyrosine (pTyr), Dok-1, Dok-2 or Csk. h WT or Dok1 −/− Dok2 −/− BMDMs were stimulated or not for 30 s with biotinylated CD200R1 mAb OX-110 and streptavidin. Cell lysates were probed with α-pLyn (Tyr 507) or α-Lyn Abs (top). A quantification of multiple independent experiments is shown at the bottom, ( n = 3). Data are from three ( b , e , g , h ) or four d independent experiments, two (pY 297 peptide) and three (pY 286 peptide) ( c ) independent experiments. Each symbol represents one mouse ( b , d , e ). Data are mean ± s.e.m. Statistical analysis: two-way ANOVA test, with multiple comparisons ( b , d , e , h ); One-way ANOVA test, with multiple comparisons ( f ). ns not significant. See also Supplementary Figs. , and Supplementary Table .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Expressing, Mass Spectrometry, Phospho-proteomics, Activation Assay, Western Blot, Quantitative Proteomics, Transduction, Knockdown, Immunoprecipitation

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200 (red curves; top) and CD47 (lavender curves; bottom) expression on J558, A20 and WEHI-231 cells (left). Filled curves, Ctrl mAbs. The right panel shows relative expression levels of CD200 and CD47. b As per Fig. , except that mAbs were used in combination: CD200 mAb OX-90 (rat IgG2a), SIRPα mAb 27 (mouse IgG2a, Fc-silent), Ctrl mAb 2A3 (rat IgG2a), and Ctrl mAb MOPC21 (mouse IgG2a, Fc-silent), ( n = 3). c – f Luciferase + Tac + GFP + WEHI-231 cells were injected intravenously into Rag1 −/− mice, followed by intraperitoneal injection of Tac mAb combined with the indicated mAbs every 2 days starting from day 4, ( n = 5). c Schematic representation of the experimental workflow. Tumor progression was measured over time using luminescence. Representative photographs of mice ( d ) and quantification ( e ). f Kaplan – Meier curves of survival. g Phagocytosis of normal activated human T cells or B cells by autologous human macrophages, in the presence of samalizumab (human IgG1, Fc-silent), CD47 mAb B6H12 (human IgG1, Fc-silent) or Ctrl mAb (human IgG1, Fc-silent). T cells were not opsonized (implying phagocytosis was mediated by SLAMF7), whereas B cells were opsonized with rituximab (human IgG1, Fc-active) ( n = 3). Data are from three ( a , b , g ) or two ( c – f ) independent experiments. Each symbol represents one mouse or donor. Data are mean ± s.e.m. Statistical analysis: two-way ANOVA test, with multiple comparisons ( b , e ); log-rank (Mantel-Cox) test ( f ); one-way ANOVA test, with multiple comparisons ( g ). ns, not significant.

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Expressing, Luciferase, Injection

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Treatment of Wlds mice with blocking anti-CD200 antibody results in worsened EAE with increased macrophage/microglia infiltrates in the CNS. After the induction of EAE, Wlds and WT mice were treated with 200 μg/100 μl of blocking anti-CD200 antibody injected intravenously every other day from days 10 to 20. Control WT and Wlds mice were treated with PBS alone. Eight mice per treatment group were evaluated. a: Wlds mice treated with anti-CD200 antibody experienced a more severe disease course than untreated Wlds mice (P < 0.05, Student’s t-test—area under the curve). In comparison, disease in WT mice was similar even after treatment with anti-CD200 antibody (P = NS, Student’s t-test). b: Spinal cord sections harvested at day 20 from treated and control mice demonstrate enhanced immunofluorescence staining of macrophages/microglia (white arrows) in the CNS of anti-CD200-treated Wlds mice compared with Wlds controls. Macrophage/microglia staining was similar in treated and untreated WT mice. Immunofluorescence staining demonstrates more SMI-32-positive axonal ovoids (white arrows) in the spinal cord white matter of treated Wlds mice, compared with untreated controls. c and d: We performed flow cytometric analysis of immune cell populations in the spinal cords isolated from WT and Wlds mice treated with anti-CD200 antibody or rat IgG control antibody (days 10 to 20) on day 20 after immunization. The results from three to four mice per group were averaged and are shown in table form in d. Also shown is a representative FACS analysis of spinal cords from WT and Wlds mice treated with control Ig or anti-CD200 antibody and stained with CD11b-phycoerythrin and CD45-allophycocyanine (APC) antibodies (c). Original magnifications, ×10.

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Blocking Assay, Injection, Control, Comparison, Immunofluorescence, Staining, Isolation

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Decreased ubiquitination of CD200 in spinal cord lysates of Wlds mice. a: Representative immunoblot of spinal cord lysates from naïve WT mice (lanes 1 and 2), naïve Wlds mice (lanes 3 and 4), WT mice day 22 after immunization (lanes 5 and 6), Wlds mice day 22 after immunization (lanes 7 and 8), WT mice day 60 after immunization (lanes 9 and 10), and Wlds mice day 60 after immunization (lanes 11 and 12) shows increased expression of CD200 in Wlds spinal cord lysates at all time points from compared with those from WT mice. β-Actin control immunoblot shows similar protein amounts in all samples. b: Densitometric quantification of immunoblots demonstrates increased expression of CD200 during the course of EAE in Wlds mice but not WT mice. c: Immunoprecipitation of CD200, with immunoblotting (IB) of ubiquitin and CD200. Sample numbers are the same as in a, except sample 10 was omitted. There was decreased expression ubiquitination of CD200 in Wlds mice samples at d0 and d22 compared with WT samples. At d60, the expression of ubiquitin was increased in Wlds samples and was comparable with the WT sample.

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Control, Immunoprecipitation

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Increased expression of CD200 in the spinal cord of Wlds mice. Spinal cord sections from WT and Wlds mice on days 0, 22, and 60 after immunization were double-stained with CD200 (green) and NeuN (red) marker for neurons. a: Shown are representative merged confocal images. CD200 expression is markedly increased in Wlds sections compared with WT sections, with increasing expression after the induction of EAE. b and c: Splitway confocal images showing co-localization of CD200 and NeuN staining in WT (b) and Wlds (c) sections. CD200 expression is increased on Wlds neuronal bodies and processes. d: Confocal merge profiles and intensity profile shows co-localization of CD200 (green) and NeuN (red) in the surface and cytoplasm of cells and processes but not the nucleus. e: Confocal intensity profile of CD200 staining shows a punctate pattern of staining consistent with surface staining of the molecule. f: Confocal reconstruction (2.5-dimension) of Z-stacked images demonstrates punctate areas of high-intensity staining (red > yellow > green), consistent with surface staining (red), as well as medium intensity staining in cytoplasmic regions (yellow). Original magnifications, ×63.

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Expressing, Staining, Marker

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Increased expression of CD200 during EAE co-localizes with CNPase and GFAP marker. a: Splitway confocal images show partial co-localization of CD200 and CNPase markers in Wlds and WT spinal cord sections. Expression in both strains is enhanced at day 22 after immunization compared with naïve spinal cords. b: Splitway confocal images show partial co-localization of CD200 and GFAP markers in Wlds and WT spinal cord sections. Expression is enhanced particularly in Wlds sections at day 22 after immunization compared with naïve spinal cords. Original magnifications, ×63.

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Expressing, Marker

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Expression of CD200 in Splenocytes from Wld s Mice

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Expressing

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Neuronal cultures from Wlds E16 embryos are protected from LPS-activated microglial-induced toxicity. Cortical neuronal cultures were derived from WT and Wlds E16 embryos and plated at a high-density concentration of 200,000 cells/well/0.5 ml in 24-well plates. a: Representative fluorescence microscopy photomicrographs of MAP-2 (red), CD200 (green), and merged images from cortical cultures. CD200 expression is increased in Wlds cultures compared with WT cultures, and co-localizes with MAP-2-positive cells (white arrows). In some cases, CD200 expression does not co-localize with MAP-2 (arrowheads). Controls are stained with isotype control antibody and secondary antibodies. b and c: Shown are representative photomicrographs of WT and Wlds neuronal cultures with LPS-activated (b) or IFN-γ-activated (c) primary microglia. Cultures were immunostained with anti-MAP-2 antibody (red) and LB4 (green). Wlds axons and neurons remain intact after co-culture with activated microglia; however, there is significant increase in axonal beading in WT co-cultures. Percentage of beaded axons/total number of axons in 10 fields was quantified for each condition. Protection of Wlds neurons from neurotoxicity induced by activated microglia is ameliorated after the addition of a blocking anti-CD200 antibody or anti-CD200 F(Ab′)2 fragment (both conditions, P < 0.0001; Student’s t-test). Original magnifications, ×40 (a); ×63 (b, c).

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Derivative Assay, Concentration Assay, Fluorescence, Microscopy, Expressing, Staining, Control, Co-Culture Assay, Blocking Assay

Journal: International journal of cosmetic science

Article Title: Mechanical epilation exerts complex biological effects on human hair follicles and perifollicular skin: An ex vivo study approach.

doi: 10.1111/ics.12923

Figure Lengend Snippet: FIGURE 8 Hair follicle (HF) immune privilege is transiently perturbed by epilation. (a, b) Quantitative immuno-histomorphometry of MHC class I, depicted as fold change of relative staining intensity, normalized to the respective control group of each day, in the hair bulb (a) and bulge (b) and CD200 expression, depicted as fold of relative staining intensity, normalized to the respective control group of each day in the bulge (c) at days 0, 3, and 6 post-epilation. The relative staining intensity of each protein was measured in the specific reference area of the sections. N = 2–15 HFs/group from one independent experiment (one donor, two punches per experimental group). Mean ± SEM. Data were analysed with a D'Agostino & Pearson normality test. If data were normally distributed, an unpaired Student's t-test was used (CD200); if data were not normally distributed, a Mann–Whitney U test was used (MHCI). Unpaired Student's t-test ##p < 0.01, Mann–Whitney U test, *p < 0.05. Representative images of MHC class I and CD200 immunostainings, depicting the evaluation areas. DP, dermal papilla; HM, hair matrix; HS, hair shaft; ORS, outer root sheath. Scale bar, 50 μm.

Article Snippet: Antigen Primary antibody Secondary antibody Detection systemHost Manufacturer Dilution Type Manufacturer CD200 Mouse Serotec 1:250 Biotinylated goat anti-mouse Beckman Coulter TSA-FITC CK15 Mouse Chemicon 1:400 Biotinylated goat anti-mouse Beckman Coulter TSA-FITC ICAM-1 Mouse AbD Serotech 1:750 Biotinylated goat anti-mouse Beckman Coulter AEC Substrate Kit, Peroxidase (HRP) MHC class I Mouse DAKO 1:1500 Biotinylated goat anti-mouse Beckman Coulter TSA-rhodamine MHC class II Mouse DAKO 1:50 Rhodamine-labelled goat anti-mouse Jackson ImmunoResearch NGF Goat Santa Cruz 1:100 Biotinylated rabbit anti-goat Vector laboratories TSA-FITC NK1R Rabbit Abcam 1:100 Polymer-labelled HRP anti-rabbit EnVision® + System– HRP Kit, DAKO (Agilent Technologies) TRPV1/ TRPA1 Rabbit Thermo Fisher Scientific 1:50 FITC-conjugated goat anti-rabbit Jackson ImmunoResearch IF amplification: anti-fluorescein/ oregon green polyclonal (goat) antibody Alexa Fluor® 488, Invitrogen, 1:100 Abbreviations: AEC, 3-amino-9 ethylcarbazole; FITC, fluorescein isothiocyanate; TSA, Tyramide signal amplification. medulla [59–61], while most of the pigmented anagen hair bulbs remained macroscopically visible in the subcutis (Figure 2a).

Techniques: Staining, Control, Expressing, MANN-WHITNEY