Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Antibodies.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Purification

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Antibody cocktail for lung DC staining.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Staining

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Flow cytometric analyses of human lung DC subsets. As depicted, human lung samples are pregated on single, live, CD45 + cells. The CD45 + population of the lung then is further gated on CD3 − and CD16/CD19/CD20 − cells (also called LIN − ), before gating on all HLA‐DR + cells. From here, monocytes are excluded by gating on CD88 − cells only and CD14 − CD88 − and CD14 + CD88 − are used for subsequent gating on the different DC subsets. Within the CD88 − population, pDC can be identified as CD123 + CD169 − cells and pre‐DC as CD123 int/+ CD169 + cells. Subsequently, the CD123/CD169 double negative population is further gated on CD141 + cDC1 (optional also CADM1 + ) or CD1c + cells, which contain a mixture of cDC2 (DC2 and DC3). These CD1c + cDC2 can be subdivided using CD5 and CD14 and by gating on the CD5 + CD14 − DC2, CD5 − CD14 − DC3, and CD5 − CD14 + DC3 subsets. Samples shown here were acquired on a CYTEK Aurora 5L.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques:

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Summary of marker expression on analyzed cell populations.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Marker, Expressing

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Reagents and antibodies used for flow cytometric analysis.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques:

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Staining workflow for human skin DC panel.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Staining, Incubation, Blocking Assay

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Gating strategy for flow cytometry analysis of human skin. The skin was enzymatically digested to generate single‐cell suspensions. (A) Gating strategy for viable CD45 + cells after exclusion of cellular debris, doublets, and dead cells. (B) CD3 + T cells and CD19 + B cells as well as CD14 + cells were excluded before DC characterization. (C) HLA‐DR expressing cells comprise DC, which can be further subdivided into CD141 + CD1c + cDC1 and CD1c + subsets, consisting of CD207 + LC and CD207 − cDC2. (D) Surface expression of co‐stimulatory markers CD40, CD80, and CD86 on the indicated DC subsets: cDC1 (orange), cDC2 (grey), and LC (blue). MFI values are shown. cDC, conventional DC; LC, Langerhans cells.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Flow Cytometry, Expressing

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Summary of marker expression on skin DC populations.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Marker, Expressing

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Reagents, antibodies, chemicals, and solutions.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Staining

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Dilution of antibodies used for flow cytometry.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Cytometry

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Gating strategy for the characterization of dendritic cell subsets in human gingiva. (i) Cells are pregated according to their size and granularity (FSC‐A/SSC‐A) to exclude debris and dead cells. (ii, iii) Doublets are excluded by gating on the FSC and SSC area (A) and height (H). (iv) Red blood cells are excluded by using SSC‐B‐H and SSC‐H. (v) Dead cells are excluded using Zombie staining. (vi) CD45 + hematopoietic cells are selected. (vii) CD3 + T cells and CD19 + B cells are excluded. (viii) CD66b + neutrophils and CD56 + NK cells are excluded. (ix) HLA‐DR + cells are selected. (x) CD123 + , CD45RA + , and CD123‐CD45RA +∖− cells are further gated, which represent the pre‐DC, pDC, and DC subpopulations, respectively. (xi) CD5 + AXL + cells represent the pre‐DC subpopulation and CD5‐AXL‐ cells constitute the pDC subpopulation. (xii) LC cells are isolated from other DC cells by their expression of both CD207 and Epcam. (xiii) Conventional DC (cDC) are divided into cDC1 and cDC2 according to their CD141 and CD1c expression. While cDC1 are positive for CD141 and negative for CD1c, cDC2 are positive for CD1a and negative for CD141. Data acquisition was performed on a Cytek Aurora Flow Cytometry System and data was analyzed using FlowJo V10.8.1 software.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Staining, Isolation, Expressing, Flow Cytometry, Software

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Summary of marker expression on analyzed cell populations.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Marker, Expressing

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Used antibodies.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques:

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Summary of marker expression on analyzed cell populations.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Marker, Expressing

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Reagents and antibodies use for flow cytometric analysis.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques:

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Staining workflow for human tumor cDC panel.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Staining, Incubation

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Proposed tumor DC staining panel.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Staining, Marker

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Summary of marker expression on analyzed cell populations.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Marker, Expressing

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Reagents and antibodies used for flow cytometric analysis

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques:

Journal: European Journal of Immunology

Article Title: Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC

doi: 10.1002/eji.202250325

Figure Lengend Snippet: Staining workflow for human TDLN cDC panel.

Article Snippet: CD1c , PE , AD5‐8E7 , Miltenyi , 130‐110‐536 , 1:100.

Techniques: Staining, Incubation

Journal: Inflammatory Bowel Diseases

Article Title: CD1a-Expressing Monocytes as Mediators of Inflammation in Ulcerative Colitis

doi: 10.1093/ibd/izy073

Figure Lengend Snippet: Incubation of PBMCs with PC affects frequencies of monocytes, macrophages, and CD4 T cells. A, Flow cytometric analysis of PBMCs from UC patients (n = 13) and non-UC donors (n = 28) that were incubated in the absence (control n = 39) or presence of PC (n = 41) and PHA (n = 40) for 48 hours depicted as boxplot diagrams. B, Cell trace CFSE dilution of CD14+ CD1a+ monocytes. C, Correlation analysis of frequencies of CD14+ CD1a+ monocytes and Th1 of Th2 cells depicted as scatter plots. The method was Pearson; numbers display correlation coefficients (cor-values) and P values. D, Flow cytometric analysis of CD4+ T cells for intracellular expression of IFNγ, IL-4, or IL-17 depicted as boxplots. PBMCs (n = 4) were incubated in the absence or presence of PC and anti-CD1a antibody. For comparison of groups, ANOVA followed by Tukey’s HSD was conducted. Boxes represent upper and lower quartiles, whiskers represent variability, and outliers are plotted as individual points ( *** 0.001; ** 0.01; *0.05).

Article Snippet: To analyze the effect of an anti-CD1a antibody on cells stimulated with PC, cells were incubated for 1 hour with 10 μg/mL of anti-human CD1a (Bio X Cell, West Lebanon, USA) and 10 μg/mL of a corresponding isotype antibody (Biolegend, San Diego, CA, USA) before 200 μg/mL of PC (Sigma-Aldrich, St. Louis, USA) was added.

Techniques: Incubation, Control, Expressing, Comparison

Journal: Inflammatory Bowel Diseases

Article Title: CD1a-Expressing Monocytes as Mediators of Inflammation in Ulcerative Colitis

doi: 10.1093/ibd/izy073

Figure Lengend Snippet: Anti-CD1a antibodies affect frequencies of CD4+ T cells. Flow cytometric analysis of PBMCs from 2 different donors that were incubated in triplicate for 5 days with PC in the presence (n = 6) or absence of anti-CD1a antibodies (n = 6) or PHA (n = 6) depicted as boxplot diagrams. For comparison of groups, ANOVA followed by Tukey’s HSD was conducted. Boxes represent upper and lower quartiles, whiskers represent variability, and outliers are plotted as individual points ( *** 0.001; ** 0.01; *0.05).

Article Snippet: To analyze the effect of an anti-CD1a antibody on cells stimulated with PC, cells were incubated for 1 hour with 10 μg/mL of anti-human CD1a (Bio X Cell, West Lebanon, USA) and 10 μg/mL of a corresponding isotype antibody (Biolegend, San Diego, CA, USA) before 200 μg/mL of PC (Sigma-Aldrich, St. Louis, USA) was added.

Techniques: Incubation, Comparison

Journal: Inflammatory Bowel Diseases

Article Title: CD1a-Expressing Monocytes as Mediators of Inflammation in Ulcerative Colitis

doi: 10.1093/ibd/izy073

Figure Lengend Snippet: Ex vivo analysis of CD14+ CD1a+ monocytes for co-expression of CCR2, CD86, and TSLPR. Flow cytometric analysis of PBMCs from UC patients (n = 21) and non-UC (n = 9) donors depicted as boxplot diagrams. Boxes represent upper and lower quartiles, whiskers represent variability, and outliers are plotted as individual points. For comparison of groups, a Student t test was performed ( *** 0.001; ** 0.01; *0.05).

Article Snippet: To analyze the effect of an anti-CD1a antibody on cells stimulated with PC, cells were incubated for 1 hour with 10 μg/mL of anti-human CD1a (Bio X Cell, West Lebanon, USA) and 10 μg/mL of a corresponding isotype antibody (Biolegend, San Diego, CA, USA) before 200 μg/mL of PC (Sigma-Aldrich, St. Louis, USA) was added.

Techniques: Ex Vivo, Expressing, Comparison

Journal: Inflammatory Bowel Diseases

Article Title: CD1a-Expressing Monocytes as Mediators of Inflammation in Ulcerative Colitis

doi: 10.1093/ibd/izy073

Figure Lengend Snippet: TAG and cholesterol levels increase in the NSG-UC mouse model and correlate with CD1a-expressing monocytes. NSG-UC mice were engrafted with PBMCs derived from UC patients (n = 4), challenged with 10% ethanol at day 8 and 50% ethanol at days 15 and 18. A, TAG and cholesterol levels depicted as boxplot diagrams; nchallenged group (control, n = 12), ethanol challenged group (ethanol, n = 21). Whiskers represent variability, and outliers are plotted as individual points. B, Correlation analysis of CD14+ CD1a+ monocytes with TAG and cholesterol levels depicted as scatter plots. (TAG n = 24, cholesterol n = 25). The method was Spearman; numbers display correlation coefficients ( rho values) and P values.

Article Snippet: To analyze the effect of an anti-CD1a antibody on cells stimulated with PC, cells were incubated for 1 hour with 10 μg/mL of anti-human CD1a (Bio X Cell, West Lebanon, USA) and 10 μg/mL of a corresponding isotype antibody (Biolegend, San Diego, CA, USA) before 200 μg/mL of PC (Sigma-Aldrich, St. Louis, USA) was added.

Techniques: Expressing, Derivative Assay, Control

Journal: Inflammatory Bowel Diseases

Article Title: CD1a-Expressing Monocytes as Mediators of Inflammation in Ulcerative Colitis

doi: 10.1093/ibd/izy073

Figure Lengend Snippet: NSG-UC mice benefit from treatment with anti-CD1a antibodies. NSG mice were engrafted with PBMCs derived from UC patients (NSG-UC, n = 3) and 1 non-UC donor (NSG-non-UC). Mice were challenged with 10% ethanol at day 8 and 50% ethanol at days 15 and 18 and treated with 30 µg of anti-CD1a antibody or isotype control at days 7, 14, and 17. Control: NSG-non-UC, n = 4; NSG-UC, n = 8. Challenged control (ethanol + isotype): NSG-non-UC, n = 4; NSG-UC, n = 16. Challenged and treated with anti-CD1a antibody (ethanol + anti-CD1a): NSG-non-UC, n = 4; NSG-UC, n = 16. A, Clinical colon and histological scores of NSG-UC mice depicted as boxplot diagrams. B, Macrophotographs of colons at autopsy. a, NSG-UC unchallenged control. b, NSG-non-UC challenged control. c, NSG-UC challenged control, NSG-UC. d, Challenged and treated with anti-CD1a antibody. C, Photomicrographs of H&E-stained sections of distal parts of the colon from mice. Arrows indicate edema and influx of inflammatory cells. D, Frequencies of human leucocytes isolated from colons of mice. Samples from each group were pooled. Mean values are given; error bars are SD. Quantification was performed using flow cytometric analysis. Sample sizes: control n = 3, ethanol + isotype n = 3, ethanol + anti-CD1a n = 3. E, mRNA expression levels of mTARC, mTGFß1, HGF, and hIFNγ depicted as boxplots. Lg: delta CT, logarithmic delta cycle threshold. Boxes represent upper and lower quartiles. Whiskers represent variability, and outliers are plotted as individual points. Labels given on x-axes on the bottom row apply to all charts. For comparison of groups, ANOVA followed by Tukey’s HSD was conducted.

Article Snippet: To analyze the effect of an anti-CD1a antibody on cells stimulated with PC, cells were incubated for 1 hour with 10 μg/mL of anti-human CD1a (Bio X Cell, West Lebanon, USA) and 10 μg/mL of a corresponding isotype antibody (Biolegend, San Diego, CA, USA) before 200 μg/mL of PC (Sigma-Aldrich, St. Louis, USA) was added.

Techniques: Derivative Assay, Control, Staining, Isolation, Expressing, Comparison

Journal: Inflammatory Bowel Diseases

Article Title: CD1a-Expressing Monocytes as Mediators of Inflammation in Ulcerative Colitis

doi: 10.1093/ibd/izy073

Figure Lengend Snippet: Treatment of NSG-UC mice with anti-CD1a antibodies affects CD14+ CD1a+ monocytes. NSG mice were engrafted and treated as in . Human leucocytes were isolated from mouse spleen and subjected to flow cytometric analysis. A, Frequencies of CD14+ CD1a+ and CD11b+ CD1a+ cells depicted as boxplot diagrams. Boxes represent upper and lower quartiles. Whiskers represent variability, and outliers are plotted as individual points. B, Correlation analysis of frequencies of CD14+ CD1a+ monocytes with subsets of B and T cells cells depicted as scatter plots. The method was Spearman; numbers display correlation coefficients ( rho values) and P values.

Article Snippet: To analyze the effect of an anti-CD1a antibody on cells stimulated with PC, cells were incubated for 1 hour with 10 μg/mL of anti-human CD1a (Bio X Cell, West Lebanon, USA) and 10 μg/mL of a corresponding isotype antibody (Biolegend, San Diego, CA, USA) before 200 μg/mL of PC (Sigma-Aldrich, St. Louis, USA) was added.

Techniques: Isolation

Journal: International Journal of Oncology

Article Title: E6-regulated overproduction of prostaglandin E2 may inhibit migration of dendritic cells in human papillomavirus 16-positive cervical lesions

doi: 10.3892/ijo.2020.4983

Figure Lengend Snippet: Expression of DCs in human papillomavirus 16-positive cervical lesions. (A) CD1a and CD83 immunostaining in cervical biopsy specimens. Original magnification, ×200. Semi-quantitative evaluation of CD1a and CD83 expression was performed in normal squamous epithelium (n=4), LSIL (n=5), HSIL (n=5) and SCC (n=5). * P<0.05 vs. Normal. (B) Preliminary verification in animal experiments. Qtracker™ 705-labeled DCs were injected into control mice, CaSki-bearing mice and SiHa-bearing mice. Popliteal lymph nodes were collected and the number of labeled DCs was detected by flow cytometry. DCs, dendritic cells; HSIL, high-grade squamous intraepithelial lesion; HPF, high-power field; LSIL, low-grade squamous intraepithelial lesion; SCC, squamous carcinoma.

Article Snippet: After dewaxing and rehydration, heat-mediated antigen retrieval was performed with 1 mM EDTA (pH 9.0) in a pressure boiler for 10 min. Microsomal PGE synthase (mPGES)-1 (1:100; cat. no. 160140; Cayman Chemical Company), mPGES-2 (1:100; cat. no. 160145; Cayman Chemical Company) and cytosolic PGE synthase (cPGES) (1:100; cat. no. 18219; Cayman Chemical Company), CD83 (1:50; cat. no. bs-4826R; BIOSS) and CD1a (prediluted; 1 drop; cat. no. ZA-0544; OriGene Technologies, Inc.) were incubated with the sections overnight at 4°C.

Techniques: Expressing, Immunostaining, Labeling, Injection, Flow Cytometry