Structured Review

Becton Dickinson anti mhc class ii
Turnover of SI-LP and MLN <t>CD103</t> + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs <t>(MHC</t> class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).
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Images

1) Product Images from "Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans"

Article Title: Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20080414

Turnover of SI-LP and MLN CD103 + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs (MHC class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).
Figure Legend Snippet: Turnover of SI-LP and MLN CD103 + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs (MHC class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, Staining, Immunohistochemistry, Derivative Assay

Localization of CD103 + and CD103 − MHC class II + CD11c + cells within the small intestinal mucosa. (A and B) Expression of CD103 by DCs in small intestinal villus and SILT. (A) Images show overlays of CD103 (red) in combination with CD11c (blue) and MHC class II (green), with insets showing DAPI staining. Arrows point toward MHC class II hi CD11c + DCs coexpressing CD103. Bars, 50 μm. (B) The graph is percentage of MHC class II hi CD11c + cells in SI villus or dome region of SILT that express CD103. Results are mean and SD ( n = 5 mice per group). ***, P
Figure Legend Snippet: Localization of CD103 + and CD103 − MHC class II + CD11c + cells within the small intestinal mucosa. (A and B) Expression of CD103 by DCs in small intestinal villus and SILT. (A) Images show overlays of CD103 (red) in combination with CD11c (blue) and MHC class II (green), with insets showing DAPI staining. Arrows point toward MHC class II hi CD11c + DCs coexpressing CD103. Bars, 50 μm. (B) The graph is percentage of MHC class II hi CD11c + cells in SI villus or dome region of SILT that express CD103. Results are mean and SD ( n = 5 mice per group). ***, P

Techniques Used: Expressing, Staining, Mouse Assay

2) Product Images from "Distinct Roles of Vaccinia Virus NF-κB Inhibitor Proteins A52, B15, and K7 in the Immune Response"

Article Title: Distinct Roles of Vaccinia Virus NF-κB Inhibitor Proteins A52, B15, and K7 in the Immune Response

Journal: Journal of Virology

doi: 10.1128/JVI.00575-17

Deletion of the A52R , B15R , and K7R genes influences peritoneal cell migration. Shown are the percentages and absolute numbers of CD11c + MHC-II + dendritic cells (DCs) (A and B), Ly6G + CD11b + neutrophils (N) (C and D), NKp46 + CD3 − natural killer cells (NK) (E and F), CD19 + B cells (B) (G), F4/80 low CD11b low monocytes (M) (H), CD4 + CD3 + T cells (CD4) (I), and CD8 + CD3 + T cells (CD8) (J) in the peritoneal cavity of BALB/c mice at 6 h postinjection of 10 7 PFU of NYVAC-C or the NYVAC-C deletion mutants. Graphs show the mean ± standard error of the mean (SEM); each point represents an individual mouse. Data are representative of two independent experiments. *, P
Figure Legend Snippet: Deletion of the A52R , B15R , and K7R genes influences peritoneal cell migration. Shown are the percentages and absolute numbers of CD11c + MHC-II + dendritic cells (DCs) (A and B), Ly6G + CD11b + neutrophils (N) (C and D), NKp46 + CD3 − natural killer cells (NK) (E and F), CD19 + B cells (B) (G), F4/80 low CD11b low monocytes (M) (H), CD4 + CD3 + T cells (CD4) (I), and CD8 + CD3 + T cells (CD8) (J) in the peritoneal cavity of BALB/c mice at 6 h postinjection of 10 7 PFU of NYVAC-C or the NYVAC-C deletion mutants. Graphs show the mean ± standard error of the mean (SEM); each point represents an individual mouse. Data are representative of two independent experiments. *, P

Techniques Used: Migration, Mouse Assay

3) Product Images from "Aspirin inhibits RANKL-induced osteoclast differentiation in dendritic cells by suppressing NF-κB and NFATc1 activation"

Article Title: Aspirin inhibits RANKL-induced osteoclast differentiation in dendritic cells by suppressing NF-κB and NFATc1 activation

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-019-1500-x

Dendritic cells (DCs) have greater T cell proliferation promotion ability. a , b DCs generated from wild-type mice expressed high levels of CD11c, MHC class II, CD80, and CD86 surface molecules. c DCs induced proliferation of allogeneic CD4 + T cells in a mixed lymphocyte reaction ( P
Figure Legend Snippet: Dendritic cells (DCs) have greater T cell proliferation promotion ability. a , b DCs generated from wild-type mice expressed high levels of CD11c, MHC class II, CD80, and CD86 surface molecules. c DCs induced proliferation of allogeneic CD4 + T cells in a mixed lymphocyte reaction ( P

Techniques Used: Generated, Mouse Assay

Aspirin has no influence on DC apoptosis, proliferation, or immunophenotype. a Aspirin was not found to exert different effects on cell proliferation at the concentrations tested ( P > 0.05). b Immature DCs (imDCs) expressed low levels of CD11c, MHC class II, CD80, and CD86 surface molecules; mature DCs highly expressed these surface molecules. Expression of surface markers on DCs with or without aspirin treatment showed no obvious differences. c The addition of aspirin had no influence on DC apoptosis ( P > 0.05). All experiments are representative of three replicates. * P
Figure Legend Snippet: Aspirin has no influence on DC apoptosis, proliferation, or immunophenotype. a Aspirin was not found to exert different effects on cell proliferation at the concentrations tested ( P > 0.05). b Immature DCs (imDCs) expressed low levels of CD11c, MHC class II, CD80, and CD86 surface molecules; mature DCs highly expressed these surface molecules. Expression of surface markers on DCs with or without aspirin treatment showed no obvious differences. c The addition of aspirin had no influence on DC apoptosis ( P > 0.05). All experiments are representative of three replicates. * P

Techniques Used: Expressing

4) Product Images from "Naive tumor-specific CD4+ T cells differentiated in vivo eradicate established melanoma"

Article Title: Naive tumor-specific CD4+ T cells differentiated in vivo eradicate established melanoma

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20091921

γ c signaling on host DC is required for survival and differentiation of TRP-1 T cells in vivo. (A) Differential activation of TRP-1 CD4 + T cells in RAG −/− and RAG −/− γ c −/− hosts. Spleens from RAG −/− or RAG −/− γ c −/− mice were isolated and stained for TRP-1 CD4 + T cells. Shown are CD62L, CD122, ICOS, and CD25 expression on gated TRP-1 CD4 + T cells from indicated host. (B) IFN-γ and CXCL9 are differentially expressed in the serum at 1 wk in RAG −/− and RAG −/− γ c −/− hosts after TRP-1 CD4 + T cell transfer. Horizontal bars represent mean. (C) TRP-1 CD4 + T cells expand in RAG −/− γ c −/− hosts initially but fail to survive after 4 wk. Error bars indicate SEM. (D) Flow cytometry of IFN-γ and IL-17 expression in TRP-1 CD4 + T cells isolated from tumor bearing RAG −/− and RAG −/− γ c −/− mice 4 wk after transfer. T cells were activated with PMA and ionomycin for 4 h and then fixed and permeabilized and stained with anti–IFN-γ and IL-17 antibodies. (E) Tbet expression in TRP-1 CD4 + T cells 4 wk after transfer. (F) MHC class II, CD80, and CD40 expression in RAG −/− γ c −/− hosts. Top flow diagram indicates CD11c high MHC class II + cells; bottom flow histograms show CD80 and CD40 expression on gated CD11c high MHC class II + cells. Solid line represents RAG −/− mice treated with TRP-1 CD4 + T cells. Shaded histogram represents RAG −/− γ c −/− mice treated with TRP-1 CD4 + T cells. Data are representative of three independent experiments ( n = 5 mice/group).
Figure Legend Snippet: γ c signaling on host DC is required for survival and differentiation of TRP-1 T cells in vivo. (A) Differential activation of TRP-1 CD4 + T cells in RAG −/− and RAG −/− γ c −/− hosts. Spleens from RAG −/− or RAG −/− γ c −/− mice were isolated and stained for TRP-1 CD4 + T cells. Shown are CD62L, CD122, ICOS, and CD25 expression on gated TRP-1 CD4 + T cells from indicated host. (B) IFN-γ and CXCL9 are differentially expressed in the serum at 1 wk in RAG −/− and RAG −/− γ c −/− hosts after TRP-1 CD4 + T cell transfer. Horizontal bars represent mean. (C) TRP-1 CD4 + T cells expand in RAG −/− γ c −/− hosts initially but fail to survive after 4 wk. Error bars indicate SEM. (D) Flow cytometry of IFN-γ and IL-17 expression in TRP-1 CD4 + T cells isolated from tumor bearing RAG −/− and RAG −/− γ c −/− mice 4 wk after transfer. T cells were activated with PMA and ionomycin for 4 h and then fixed and permeabilized and stained with anti–IFN-γ and IL-17 antibodies. (E) Tbet expression in TRP-1 CD4 + T cells 4 wk after transfer. (F) MHC class II, CD80, and CD40 expression in RAG −/− γ c −/− hosts. Top flow diagram indicates CD11c high MHC class II + cells; bottom flow histograms show CD80 and CD40 expression on gated CD11c high MHC class II + cells. Solid line represents RAG −/− mice treated with TRP-1 CD4 + T cells. Shaded histogram represents RAG −/− γ c −/− mice treated with TRP-1 CD4 + T cells. Data are representative of three independent experiments ( n = 5 mice/group).

Techniques Used: In Vivo, Activation Assay, Mouse Assay, Isolation, Staining, Expressing, Flow Cytometry, Cytometry

Mechanism of treatment by TRP-1 CD4 + T cells. (A) Confocal microscopy of day-14 tumors, 1 wk after adoptive cell transfer with 2 × 10 5 naive TRP-1 CD4 + T cells. Tumors were frozen in OCT, cut, and stained with DAPI (blue) and MHC class II (red). Samples were analyzed by confocal microscopy (Olympus) with a 20× oil immersion objective. Bars, 50 µm. (B) 7-d tumor-bearing MHC class II −/− mice were irradiated with 5 Gy or not irradiated and treated with 2 × 10 5 naive TRP-1 CD4 + T cells. (C) 7-d tumor-bearing non-TCR Tg Tyrp1 B-w RAG −/− mice were irradiated with 5 Gy or not irradiated and treated with 2 × 10 5 naive TRP-1 CD4 + T cells. (D) 11-d tumor-bearing RAG −/− mice were treated with naive TRP-1 CD4 + T cells or not and, in addition, some treated mice received one injection of 500 µg of neutralizing anti–IFN-γ antibodies on day 7 of ACT. Data are representative of four independent experiments with five to eight mice per group. (E) DC activation in lymphopenic mice after ACT with TRP-1 CD4 + T cells. Flow cytometry of CD11c high MHC class II high and CD86 high DCs from tumor-bearing RAG −/− mice undergoing treatment or not. Bar graphs indicate absolute numbers of CD11c high MHC class II + CD86 high cells. Values represent SEM ( n = 3 mice/group; **, P
Figure Legend Snippet: Mechanism of treatment by TRP-1 CD4 + T cells. (A) Confocal microscopy of day-14 tumors, 1 wk after adoptive cell transfer with 2 × 10 5 naive TRP-1 CD4 + T cells. Tumors were frozen in OCT, cut, and stained with DAPI (blue) and MHC class II (red). Samples were analyzed by confocal microscopy (Olympus) with a 20× oil immersion objective. Bars, 50 µm. (B) 7-d tumor-bearing MHC class II −/− mice were irradiated with 5 Gy or not irradiated and treated with 2 × 10 5 naive TRP-1 CD4 + T cells. (C) 7-d tumor-bearing non-TCR Tg Tyrp1 B-w RAG −/− mice were irradiated with 5 Gy or not irradiated and treated with 2 × 10 5 naive TRP-1 CD4 + T cells. (D) 11-d tumor-bearing RAG −/− mice were treated with naive TRP-1 CD4 + T cells or not and, in addition, some treated mice received one injection of 500 µg of neutralizing anti–IFN-γ antibodies on day 7 of ACT. Data are representative of four independent experiments with five to eight mice per group. (E) DC activation in lymphopenic mice after ACT with TRP-1 CD4 + T cells. Flow cytometry of CD11c high MHC class II high and CD86 high DCs from tumor-bearing RAG −/− mice undergoing treatment or not. Bar graphs indicate absolute numbers of CD11c high MHC class II + CD86 high cells. Values represent SEM ( n = 3 mice/group; **, P

Techniques Used: Confocal Microscopy, Staining, Mouse Assay, Irradiation, Injection, Activated Clotting Time Assay, Activation Assay, Flow Cytometry, Cytometry

5) Product Images from "Requirement for BAFF and APRIL during B Cell Development in GALT"

Article Title: Requirement for BAFF and APRIL during B Cell Development in GALT

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1000146

BAFF-binding to PB monocytes. Flow cytometric staining of PBL with rBAFF and anti–MHC-II ( A ); anti-CD11b; and anti-CD14 ( B ). C , Top panels , Flow cytometric analysis of MHC-II + cells for CD44 expression. CD44 hi and CD44 lo subsets indicated by vertical dashed line. Bottom panels , Analysis of CD44 lo cells for L chain expression ( left panel ) and CD44 hi cells for CD14 and CD9 expression ( middle and right panels ). D , Flow cytometric staining of CD44 hi and CD44 lo subsets of MHC-II + cells with rBAFF. Shaded histogram indicates isotype control.
Figure Legend Snippet: BAFF-binding to PB monocytes. Flow cytometric staining of PBL with rBAFF and anti–MHC-II ( A ); anti-CD11b; and anti-CD14 ( B ). C , Top panels , Flow cytometric analysis of MHC-II + cells for CD44 expression. CD44 hi and CD44 lo subsets indicated by vertical dashed line. Bottom panels , Analysis of CD44 lo cells for L chain expression ( left panel ) and CD44 hi cells for CD14 and CD9 expression ( middle and right panels ). D , Flow cytometric staining of CD44 hi and CD44 lo subsets of MHC-II + cells with rBAFF. Shaded histogram indicates isotype control.

Techniques Used: Binding Assay, Flow Cytometry, Staining, Expressing

6) Product Images from "Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A-deficient mice"

Article Title: Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A-deficient mice

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200421231

Morphology, JAM-A expression, and DC distribution in lymph node and skin sections. ( A ) H E staining of a lymph node section from a Jam-A –/– mouse displays normal architecture including cortical lymphoid nodules (CN) and an expanded paracortical area (PC); PC is populated by DCs, highlighted by their strong expression of MHC class II molecule (inset). ( B ) Expression of JAM-A in a lymph node section from a Jam-A +/+ mouse is evident in sinus macrophages (asterisk), high endothelial venules (arrowheads in B and C ), and DCs in the paracortex ( C , black arrow). ( D ) In normal skin, a diffuse JAM-A expression is observed in epidermal and pilar keratinocytes, in endothelial cells of dermal vessels (arrowhead), and in scattered fusate/stellate dermal cells (inset). ( E ) A skin section from a Jam-A –/– mouse illustrates the regular distribution of MHC class II + LCs; LCs display an evident dendritic morphology with fine and long dendrites, and express Langerin (insert). ( F and G ) Sections from an inguinal lymph node obtained after FITC skin painting display green-dotted FITC + cells in the paracortex that coexpress JAM-A ( F , arrow) and Langerin ( G , arrow). MHC class II + and JAM-A + cells were detected by immunoperoxidase technique as seen in A (inset), B , C , and D . Immunofluorescence staining was used to detect JAM-A + cells (red cells in F ), MHC class II + LCs (red cells in E ), and Langerin + LCs (red cells, inset in E ; G ). Magnification: ×40 in A , ×200 in F and G , ×400 in B , D , and E , and ×600 in insert in A , C , insert in D , and insert in E . Scale bars: 500 μm in A , 50 μm in B , 20 μm in C , and 100 μm in F .
Figure Legend Snippet: Morphology, JAM-A expression, and DC distribution in lymph node and skin sections. ( A ) H E staining of a lymph node section from a Jam-A –/– mouse displays normal architecture including cortical lymphoid nodules (CN) and an expanded paracortical area (PC); PC is populated by DCs, highlighted by their strong expression of MHC class II molecule (inset). ( B ) Expression of JAM-A in a lymph node section from a Jam-A +/+ mouse is evident in sinus macrophages (asterisk), high endothelial venules (arrowheads in B and C ), and DCs in the paracortex ( C , black arrow). ( D ) In normal skin, a diffuse JAM-A expression is observed in epidermal and pilar keratinocytes, in endothelial cells of dermal vessels (arrowhead), and in scattered fusate/stellate dermal cells (inset). ( E ) A skin section from a Jam-A –/– mouse illustrates the regular distribution of MHC class II + LCs; LCs display an evident dendritic morphology with fine and long dendrites, and express Langerin (insert). ( F and G ) Sections from an inguinal lymph node obtained after FITC skin painting display green-dotted FITC + cells in the paracortex that coexpress JAM-A ( F , arrow) and Langerin ( G , arrow). MHC class II + and JAM-A + cells were detected by immunoperoxidase technique as seen in A (inset), B , C , and D . Immunofluorescence staining was used to detect JAM-A + cells (red cells in F ), MHC class II + LCs (red cells in E ), and Langerin + LCs (red cells, inset in E ; G ). Magnification: ×40 in A , ×200 in F and G , ×400 in B , D , and E , and ×600 in insert in A , C , insert in D , and insert in E . Scale bars: 500 μm in A , 50 μm in B , 20 μm in C , and 100 μm in F .

Techniques Used: Expressing, Staining, Immunofluorescence

7) Product Images from "Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans"

Article Title: Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20080414

Turnover of SI-LP and MLN CD103 + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs (MHC class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).
Figure Legend Snippet: Turnover of SI-LP and MLN CD103 + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs (MHC class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, Staining, Immunohistochemistry, Derivative Assay

Localization of CD103 + and CD103 − MHC class II + CD11c + cells within the small intestinal mucosa. (A and B) Expression of CD103 by DCs in small intestinal villus and SILT. (A) Images show overlays of CD103 (red) in combination with CD11c (blue) and MHC class II (green), with insets showing DAPI staining. Arrows point toward MHC class II hi CD11c + DCs coexpressing CD103. Bars, 50 μm. (B) The graph is percentage of MHC class II hi CD11c + cells in SI villus or dome region of SILT that express CD103. Results are mean and SD ( n = 5 mice per group). ***, P
Figure Legend Snippet: Localization of CD103 + and CD103 − MHC class II + CD11c + cells within the small intestinal mucosa. (A and B) Expression of CD103 by DCs in small intestinal villus and SILT. (A) Images show overlays of CD103 (red) in combination with CD11c (blue) and MHC class II (green), with insets showing DAPI staining. Arrows point toward MHC class II hi CD11c + DCs coexpressing CD103. Bars, 50 μm. (B) The graph is percentage of MHC class II hi CD11c + cells in SI villus or dome region of SILT that express CD103. Results are mean and SD ( n = 5 mice per group). ***, P

Techniques Used: Expressing, Staining, Mouse Assay

8) Product Images from "Long-Term Persistence of Functional Thymic Epithelial Progenitor Cells In Vivo under Conditions of Low FOXN1 Expression"

Article Title: Long-Term Persistence of Functional Thymic Epithelial Progenitor Cells In Vivo under Conditions of Low FOXN1 Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0114842

Thymus formation upon Cre-mediated reversion of the Foxn1 R allele in R/−; CreERt2 mice. 3–4 months old R/−; CreERt2 mice were injected with 4OHT at the doses shown. Images show immunohistochemical analysis of thymi or thymic rudiments from 4OHT mice 7 weeks post-injection or from a 6 week old C57BL/6 wild type (WT) control. Staining for ( A ) pan-cytokeratin (PANK; green) and PLET1 (red). Scale bars, 150 µm. ( B ) K5 (green) and K8 (red). Scale bars, 300 µm. ( C ) PANK (green) and UEA-1 (red). Scale bars, 150 µm. ( D ) PANK (green) and MHC Class II (MHCII, red). Scale bars, 150 µm. Arrowheads in ( A ) and ( B ) indicate areas of undifferentiated thymic rudiment. DAPI reveals nuclei (blue) in panels ( A–D ). ( E ) PANK (green) and CD45 (red). Scale bars 100 µm. Note that CD45 + cells are found associated with but not within the epithelium in thymic rudiments from carrier only and 0.25 µg 4OHT injected mice. 0.25 mg and 0.5 mg 4OHT injected mice and WT controls, n > 3. 1.5 mg and 2 mg 4OHT injected mice, n = 1 for each condition; equivalent data were obtained from mice injected with 1.0 mg 4OHT (n = 3).
Figure Legend Snippet: Thymus formation upon Cre-mediated reversion of the Foxn1 R allele in R/−; CreERt2 mice. 3–4 months old R/−; CreERt2 mice were injected with 4OHT at the doses shown. Images show immunohistochemical analysis of thymi or thymic rudiments from 4OHT mice 7 weeks post-injection or from a 6 week old C57BL/6 wild type (WT) control. Staining for ( A ) pan-cytokeratin (PANK; green) and PLET1 (red). Scale bars, 150 µm. ( B ) K5 (green) and K8 (red). Scale bars, 300 µm. ( C ) PANK (green) and UEA-1 (red). Scale bars, 150 µm. ( D ) PANK (green) and MHC Class II (MHCII, red). Scale bars, 150 µm. Arrowheads in ( A ) and ( B ) indicate areas of undifferentiated thymic rudiment. DAPI reveals nuclei (blue) in panels ( A–D ). ( E ) PANK (green) and CD45 (red). Scale bars 100 µm. Note that CD45 + cells are found associated with but not within the epithelium in thymic rudiments from carrier only and 0.25 µg 4OHT injected mice. 0.25 mg and 0.5 mg 4OHT injected mice and WT controls, n > 3. 1.5 mg and 2 mg 4OHT injected mice, n = 1 for each condition; equivalent data were obtained from mice injected with 1.0 mg 4OHT (n = 3).

Techniques Used: Mouse Assay, Injection, Immunohistochemistry, Staining

9) Product Images from "Divergent Effects of Mycobacterial Cell Wall Glycolipids on Maturation and Function of Human Monocyte-Derived Dendritic Cells"

Article Title: Divergent Effects of Mycobacterial Cell Wall Glycolipids on Maturation and Function of Human Monocyte-Derived Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042515

Maturation markers on DCs exposed to LPS and/or ManLAM and PIM from H37Rv. Immature DCs were stimulated with mycobacterial glycolipids (ManLAM and PIM at the concentration of 10 µg/ml and 5 µg/ml, respectively) or/and LPS (100 ng/ml) as indicated in the diagrams. After 48 h cells were harvested, stained for maturation markers CD80, CD86, and MHC class II and analyzed by flow cytometry. For each treatment mean fluorescence intensities (MFI) were related to the levels obtained for stimulation with LPS and expressed as %. The median percentage change in MFI is shown as a line. The box defines the 75th and 25th percentiles and the whiskers define the maximum and minimum values of 7–11 donors/group. The horizontal, dashed lines represent surface marker expression obtained for LPS treated DC. Groups significantly different from LPS-treated control are labeled with asterisks. Vertical bars designate significant differences between treatment groups. Wilcoxon matched pair test was used to assess statistical significance (*P
Figure Legend Snippet: Maturation markers on DCs exposed to LPS and/or ManLAM and PIM from H37Rv. Immature DCs were stimulated with mycobacterial glycolipids (ManLAM and PIM at the concentration of 10 µg/ml and 5 µg/ml, respectively) or/and LPS (100 ng/ml) as indicated in the diagrams. After 48 h cells were harvested, stained for maturation markers CD80, CD86, and MHC class II and analyzed by flow cytometry. For each treatment mean fluorescence intensities (MFI) were related to the levels obtained for stimulation with LPS and expressed as %. The median percentage change in MFI is shown as a line. The box defines the 75th and 25th percentiles and the whiskers define the maximum and minimum values of 7–11 donors/group. The horizontal, dashed lines represent surface marker expression obtained for LPS treated DC. Groups significantly different from LPS-treated control are labeled with asterisks. Vertical bars designate significant differences between treatment groups. Wilcoxon matched pair test was used to assess statistical significance (*P

Techniques Used: Concentration Assay, Staining, Flow Cytometry, Cytometry, Fluorescence, Marker, Expressing, Labeling

10) Product Images from "Exosomes from antigen-pulsed dendritic cells induce stronger antigen-specific immune responses than microvesicles in vivo"

Article Title: Exosomes from antigen-pulsed dendritic cells induce stronger antigen-specific immune responses than microvesicles in vivo

Journal: Scientific Reports

doi: 10.1038/s41598-017-16609-6

Exosomes and microvesicles from OVA-pulsed dendritic cells (DC) display similar levels of immunorelevant markers and tetraspanins. Using polystyrene-latex coated with anti-MHC class II antibodies, vesicles corresponding to the same total protein amounts were captured and analysed by flow cytometry. Quantified expression levels in mean fluorescence intensity of MHC class I and II, the adhesion molecule CD54, costimulatory CD86, CD80 and CD40 as well as the tetraspanins CD9, CD63 and CD81. All levels are MFI ratio of each marker normalised to isotype controls, conducted on four batches of exosomes (Exo) and microvesicles (MV) from OVA-pulsed (OVA) or un-pulsed DCs (UN).
Figure Legend Snippet: Exosomes and microvesicles from OVA-pulsed dendritic cells (DC) display similar levels of immunorelevant markers and tetraspanins. Using polystyrene-latex coated with anti-MHC class II antibodies, vesicles corresponding to the same total protein amounts were captured and analysed by flow cytometry. Quantified expression levels in mean fluorescence intensity of MHC class I and II, the adhesion molecule CD54, costimulatory CD86, CD80 and CD40 as well as the tetraspanins CD9, CD63 and CD81. All levels are MFI ratio of each marker normalised to isotype controls, conducted on four batches of exosomes (Exo) and microvesicles (MV) from OVA-pulsed (OVA) or un-pulsed DCs (UN).

Techniques Used: Flow Cytometry, Cytometry, Expressing, Fluorescence, Marker

Splenocytes from all mice injected with OVA-loaded exosomes (Exo-OVA), and majority of those injected with microvesicles (MV-OVA) are responsive to antigenic restimulation. Splenocytes from mice immunised with either Exo-OVA, MV-OVA or a combination of the two were restimulated in an IFNγ ELISPOT. Numbers of IFNγ-producing units were counted after restimulation with the MHC class II-associated OVA 323–339 peptide ( a ), the MHC class I-associated OVA peptide SIINFEKL ( b ), or with whole OVA protein ( c ). Control groups were immunised with vesicles without antigen (Exo-UN, MV-UN) or PBS. Results are pooled from at least three independent experiments (n = 4–7 per group per experiment). *Indicates p
Figure Legend Snippet: Splenocytes from all mice injected with OVA-loaded exosomes (Exo-OVA), and majority of those injected with microvesicles (MV-OVA) are responsive to antigenic restimulation. Splenocytes from mice immunised with either Exo-OVA, MV-OVA or a combination of the two were restimulated in an IFNγ ELISPOT. Numbers of IFNγ-producing units were counted after restimulation with the MHC class II-associated OVA 323–339 peptide ( a ), the MHC class I-associated OVA peptide SIINFEKL ( b ), or with whole OVA protein ( c ). Control groups were immunised with vesicles without antigen (Exo-UN, MV-UN) or PBS. Results are pooled from at least three independent experiments (n = 4–7 per group per experiment). *Indicates p

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

11) Product Images from "Processing and Degradation of Exogenous Prion Protein by CD11c+ Myeloid Dendritic Cells In Vitro"

Article Title: Processing and Degradation of Exogenous Prion Protein by CD11c+ Myeloid Dendritic Cells In Vitro

Journal: Journal of Virology

doi: 10.1128/JVI.76.23.12259-12264.2002

Coculture of ScGT1-1 cells and DC. (A) ScGT1-1 cells, immunolabeled with anti-β-tubulin antibodies (green); (B) ScGT1-1 cells (green to yellowish) exposed to DC, ratio 1:1, immunolabeled with anti-MHC class II antibodies (red), for 48 h. Note the extensive loss of GT1-1 cells. Remnants are surrounded by clusters of DC. Scale bar, 25 μm.
Figure Legend Snippet: Coculture of ScGT1-1 cells and DC. (A) ScGT1-1 cells, immunolabeled with anti-β-tubulin antibodies (green); (B) ScGT1-1 cells (green to yellowish) exposed to DC, ratio 1:1, immunolabeled with anti-MHC class II antibodies (red), for 48 h. Note the extensive loss of GT1-1 cells. Remnants are surrounded by clusters of DC. Scale bar, 25 μm.

Techniques Used: Immunolabeling

12) Product Images from "Conjunctival macrophages act as antigen-presenting cells in the conjunctiva during the development of experimental allergic conjunctivitis"

Article Title: Conjunctival macrophages act as antigen-presenting cells in the conjunctiva during the development of experimental allergic conjunctivitis

Journal: Molecular Vision

doi:

Phenotypic characterization of cells that engulfed OVA-gold in the conjunctiva. Conjunctival samples were prepared as described in Figure 2 . Samples were evaluated immunohistochemically to examine the expression of CD11b, CD68, and MHC class II.
Figure Legend Snippet: Phenotypic characterization of cells that engulfed OVA-gold in the conjunctiva. Conjunctival samples were prepared as described in Figure 2 . Samples were evaluated immunohistochemically to examine the expression of CD11b, CD68, and MHC class II.

Techniques Used: Expressing

13) Product Images from "Fcγ Receptor Regulation of Citrobacter rodentium Infection "

Article Title: Fcγ Receptor Regulation of Citrobacter rodentium Infection

Journal: Infection and Immunity

doi: 10.1128/IAI.01493-07

Incorporation of C . rodentium or C . rodentium -IgG complexes into DCs from FcγR-deficient mice. (A) Bone marrow-derived DCs from wild-type, FcRγ −/− (γ −/−), or FcRIIB −/− (IIB −/−) mice were cocultured with FITC-conjugated C . rodentium with anti- C . rodentium IgG or control IgG for 1 h. Cells were labeled with anti-CD11c and anti-MHC class II antibodies and analyzed by flow cytometry gated on CD11c-positive cells. CR+anti-CR Ab, C . rodentium plus anti- C . rodentium antibody; CR+control IgG, C . rodentium plus control IgG; WT, wild type; FITC-CR, FITC-conjugated C . rodentium . (B) Analysis of incorporation of FITC-conjugated C . rodentium gated on CD11c-positive and MHC class II low-positive cells. Results are representative of three independent experiments.
Figure Legend Snippet: Incorporation of C . rodentium or C . rodentium -IgG complexes into DCs from FcγR-deficient mice. (A) Bone marrow-derived DCs from wild-type, FcRγ −/− (γ −/−), or FcRIIB −/− (IIB −/−) mice were cocultured with FITC-conjugated C . rodentium with anti- C . rodentium IgG or control IgG for 1 h. Cells were labeled with anti-CD11c and anti-MHC class II antibodies and analyzed by flow cytometry gated on CD11c-positive cells. CR+anti-CR Ab, C . rodentium plus anti- C . rodentium antibody; CR+control IgG, C . rodentium plus control IgG; WT, wild type; FITC-CR, FITC-conjugated C . rodentium . (B) Analysis of incorporation of FITC-conjugated C . rodentium gated on CD11c-positive and MHC class II low-positive cells. Results are representative of three independent experiments.

Techniques Used: Mouse Assay, Derivative Assay, Labeling, Flow Cytometry, Cytometry

C . rodentium -IgG complexes incorporation effectively induced DC maturation via FcγR. (A) Bone marrow-derived DCs from FcγR-deficient mice were incubated for 24 h in the absence (white) or presence of C . rodentium and control IgG (CR+control IgG) (gray) or C . rodentium and anti- C . rodentium IgG complexes (CR+anti-CR IgG) (black). Cells from wild-type (WT), FcRγ −/− (γ −/−), and FcRIIB −/− (IIB −/−) mice are shown. DCs were stained with anti-CD86 or anti-MHC class II after gating on CD11c-positive cells and analyzed by flow cytometry. Results are from one representative experiment of three independent experiments. (B) Cytokine production of TNF-α was measured by an ELISA. The mean plus standard deviation (SD) are shown for each group (three mice per group). This experiment was evaluated by ANOVA. Values that are significantly different ( P
Figure Legend Snippet: C . rodentium -IgG complexes incorporation effectively induced DC maturation via FcγR. (A) Bone marrow-derived DCs from FcγR-deficient mice were incubated for 24 h in the absence (white) or presence of C . rodentium and control IgG (CR+control IgG) (gray) or C . rodentium and anti- C . rodentium IgG complexes (CR+anti-CR IgG) (black). Cells from wild-type (WT), FcRγ −/− (γ −/−), and FcRIIB −/− (IIB −/−) mice are shown. DCs were stained with anti-CD86 or anti-MHC class II after gating on CD11c-positive cells and analyzed by flow cytometry. Results are from one representative experiment of three independent experiments. (B) Cytokine production of TNF-α was measured by an ELISA. The mean plus standard deviation (SD) are shown for each group (three mice per group). This experiment was evaluated by ANOVA. Values that are significantly different ( P

Techniques Used: Derivative Assay, Mouse Assay, Incubation, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

14) Product Images from "Lymphoma endothelium preferentially expresses Tim-3 and facilitates the progression of lymphoma by mediating immune evasion"

Article Title: Lymphoma endothelium preferentially expresses Tim-3 and facilitates the progression of lymphoma by mediating immune evasion

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20090397

Validation of an in vitro cell model for studying interactions between ECs and autologous T lymphocytes. (A, top) Schematic of the ADV-TT construct. (bottom) Transcripts of TT in ADV-TT–infected UVECs were detected by RT-PCR. ADV-GFP and PBS were included as negative controls. (B) Expression of MHC class I, MHC class II, CD86, or CD58 in freshly isolated UVECs treated with PBS (black), IFN-γ (green), or IFN-γ + ADV–Tim-3 (red). (C) UVECs were infected with ADV-TT or ADV-GFP and incubated with CFSE-labeled autologous lymphocytes. The proliferation of lymphocytes was measured by FACS. (right) CFSE profiles of lymphocytes treated with ADV-GFP or ADV-TT. Data are represented as means ± SD of triplicates. (D) BEAS and UVECs were infected with ADV-TT at an MOI of 250 and incubated with CFSE-labeled autologous lymphocytes. The proliferation of lymphocytes was measured by FACS. (right) CFSE profiles of lymphocytes treated with ADV-TT. Data are represented as means ± SD of triplicates. (E) Cell proliferation of UVECs infected with various titers of ADV-TT. Infection of ADV-TT did not lead to an increase in UVEC cell numbers compared with infection with ADV-GFP or at any of the MOIs examined. Data are represented as means ± SD of triplicates. Similar results were observed in four (B) or three (C–E) independent experiments.
Figure Legend Snippet: Validation of an in vitro cell model for studying interactions between ECs and autologous T lymphocytes. (A, top) Schematic of the ADV-TT construct. (bottom) Transcripts of TT in ADV-TT–infected UVECs were detected by RT-PCR. ADV-GFP and PBS were included as negative controls. (B) Expression of MHC class I, MHC class II, CD86, or CD58 in freshly isolated UVECs treated with PBS (black), IFN-γ (green), or IFN-γ + ADV–Tim-3 (red). (C) UVECs were infected with ADV-TT or ADV-GFP and incubated with CFSE-labeled autologous lymphocytes. The proliferation of lymphocytes was measured by FACS. (right) CFSE profiles of lymphocytes treated with ADV-GFP or ADV-TT. Data are represented as means ± SD of triplicates. (D) BEAS and UVECs were infected with ADV-TT at an MOI of 250 and incubated with CFSE-labeled autologous lymphocytes. The proliferation of lymphocytes was measured by FACS. (right) CFSE profiles of lymphocytes treated with ADV-TT. Data are represented as means ± SD of triplicates. (E) Cell proliferation of UVECs infected with various titers of ADV-TT. Infection of ADV-TT did not lead to an increase in UVEC cell numbers compared with infection with ADV-GFP or at any of the MOIs examined. Data are represented as means ± SD of triplicates. Similar results were observed in four (B) or three (C–E) independent experiments.

Techniques Used: In Vitro, Construct, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Incubation, Labeling, FACS

15) Product Images from "Migrant memory B cells secrete luminal antibody in the vagina"

Article Title: Migrant memory B cells secrete luminal antibody in the vagina

Journal: Nature

doi: 10.1038/s41586-019-1285-1

Rapid B cell recruitment into the FRT upon secondary challenge is a prerequisite for antibody secretion into vaginal lumen a-b , C57BL/6 mice were infected vaginally with TK − HSV-2 five weeks prior. Mice were given drinking water containing 4 μg/ml of FTY720 for two weeks. a , One day after secondary challenge, the number of IgG + MHC class II + cells and CD11c hi MHC class II hi cells were analyzed by flow cytometry (n=3). b , After challenge, HSV-2-specific antibodies in vaginal wash were measured by ELISA (n=5). Sample dilution for ELISA was 1:7. Data are mean ± SEM. Data are representative of two ( f ) independent experiments, or are pooled from two ( g ) independent experiments. *, p
Figure Legend Snippet: Rapid B cell recruitment into the FRT upon secondary challenge is a prerequisite for antibody secretion into vaginal lumen a-b , C57BL/6 mice were infected vaginally with TK − HSV-2 five weeks prior. Mice were given drinking water containing 4 μg/ml of FTY720 for two weeks. a , One day after secondary challenge, the number of IgG + MHC class II + cells and CD11c hi MHC class II hi cells were analyzed by flow cytometry (n=3). b , After challenge, HSV-2-specific antibodies in vaginal wash were measured by ELISA (n=5). Sample dilution for ELISA was 1:7. Data are mean ± SEM. Data are representative of two ( f ) independent experiments, or are pooled from two ( g ) independent experiments. *, p

Techniques Used: Mouse Assay, Infection, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

16) Product Images from "Reversing Endogenous Alloreactive B cell GC Responses with Anti-CD154 or CTLA-4Ig"

Article Title: Reversing Endogenous Alloreactive B cell GC Responses with Anti-CD154 or CTLA-4Ig

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/ajt.12350

Tracking the changes in phenotype of K d Tet-DP B cells at day 7 after immunization with BALB/c or BALB.B splenocytes. Increased percentages (top) of IgD − K d Tet-DP B cells expressing MHC Class II and CD86 and their mean fluorescence intensities
Figure Legend Snippet: Tracking the changes in phenotype of K d Tet-DP B cells at day 7 after immunization with BALB/c or BALB.B splenocytes. Increased percentages (top) of IgD − K d Tet-DP B cells expressing MHC Class II and CD86 and their mean fluorescence intensities

Techniques Used: Expressing, Fluorescence

17) Product Images from "MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis"

Article Title: MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-017-0915-5

MHC class II expression on inflamed vessels. Three weeks after adoptive transfer, eye cryosections were prepared and stained for MHC class II ( green ), IBA1 ( red ) or GFAP ( red ), and CD31 ( magenta ) detection. Cell nuclei were stained with Hoechst ( blue ). Each picture was chosen as representative of an experiment conducted on three or more animals. a MHC class II and IBA1 expression at the level of vasculitis. b MHC class II, CD31, and GFAP expression at the level of vasculitis
Figure Legend Snippet: MHC class II expression on inflamed vessels. Three weeks after adoptive transfer, eye cryosections were prepared and stained for MHC class II ( green ), IBA1 ( red ) or GFAP ( red ), and CD31 ( magenta ) detection. Cell nuclei were stained with Hoechst ( blue ). Each picture was chosen as representative of an experiment conducted on three or more animals. a MHC class II and IBA1 expression at the level of vasculitis. b MHC class II, CD31, and GFAP expression at the level of vasculitis

Techniques Used: Expressing, Adoptive Transfer Assay, Staining

18) Product Images from "Functional and Pathogenic Differences of Th1 and Th17 Cells in Experimental Autoimmune Encephalomyelitis"

Article Title: Functional and Pathogenic Differences of Th1 and Th17 Cells in Experimental Autoimmune Encephalomyelitis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0015531

Cytotoxic potential of Th1 and Th17 cells towards astrocytes. 2D2 MOG-specific T cells were polarized in Th1 and Th17 conditions and co-cultured in duplicates with activated astrocytes as described in methods. A. MHC class II expression on astrocytes was analysed by FACS. B. GFP-labeled astrocytes were co-cultured with Th1 or Th17 cells in the presence of isotype control or anti-MHC class II antibodies. Cells were tracked every 30 minutes by fluorescent time-lapse microscopy. Shown is the snap-shot fluorescent picture after 48 h co-culture. Magnification: 10×. C. Quantification of the fluorescent area (surviving cells) every 6 hours in the conditions shown in B. The values were normalized to that of control (astrocytes only). D. IL-17 and IFN-γ levels were measured in the supernatants at 48 h after the co-culture by ELISA. Data shown are representative of minimum 3 experiments. *p
Figure Legend Snippet: Cytotoxic potential of Th1 and Th17 cells towards astrocytes. 2D2 MOG-specific T cells were polarized in Th1 and Th17 conditions and co-cultured in duplicates with activated astrocytes as described in methods. A. MHC class II expression on astrocytes was analysed by FACS. B. GFP-labeled astrocytes were co-cultured with Th1 or Th17 cells in the presence of isotype control or anti-MHC class II antibodies. Cells were tracked every 30 minutes by fluorescent time-lapse microscopy. Shown is the snap-shot fluorescent picture after 48 h co-culture. Magnification: 10×. C. Quantification of the fluorescent area (surviving cells) every 6 hours in the conditions shown in B. The values were normalized to that of control (astrocytes only). D. IL-17 and IFN-γ levels were measured in the supernatants at 48 h after the co-culture by ELISA. Data shown are representative of minimum 3 experiments. *p

Techniques Used: Cell Culture, Expressing, FACS, Labeling, Time-lapse Microscopy, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Cytotoxic potential of Th1 and Th17 cells towards FT7.1 cells. 2D2 MOG-specific T cells were polarized in Th1 and Th17 conditions and co-cultured in duplicates with FT7.1 cells as described in methods. A. MHC class II expression on FT7.1 cells was analysed by FACS. B. GFP-labeled FT7.1 cells were co-cultured with Th1 or Th17 cells in the presence of isotype control or anti-MHC class II antibodies. Cells were tracked every 30 minutes by fluorescent time-lapse microscopy. Shown is the snap-shot fluorescent picture after 48 h co-culture. Magnification: 10×. C. Quantification of the fluorescent area (surviving cells) every 6 hours in the conditions shown in B. The values were normalized to that of control (FT7.1 cells only). D. IL-17 and IFN-γ levels were measured in the supernatants at 48 h after the co-culture by ELISA. Data shown are representative of minimum 3 experiments. ns-not significant.
Figure Legend Snippet: Cytotoxic potential of Th1 and Th17 cells towards FT7.1 cells. 2D2 MOG-specific T cells were polarized in Th1 and Th17 conditions and co-cultured in duplicates with FT7.1 cells as described in methods. A. MHC class II expression on FT7.1 cells was analysed by FACS. B. GFP-labeled FT7.1 cells were co-cultured with Th1 or Th17 cells in the presence of isotype control or anti-MHC class II antibodies. Cells were tracked every 30 minutes by fluorescent time-lapse microscopy. Shown is the snap-shot fluorescent picture after 48 h co-culture. Magnification: 10×. C. Quantification of the fluorescent area (surviving cells) every 6 hours in the conditions shown in B. The values were normalized to that of control (FT7.1 cells only). D. IL-17 and IFN-γ levels were measured in the supernatants at 48 h after the co-culture by ELISA. Data shown are representative of minimum 3 experiments. ns-not significant.

Techniques Used: Cell Culture, Expressing, FACS, Labeling, Time-lapse Microscopy, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

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Article Title: Innate immune adaptor TRIF deficiency accelerates disease progression of ALS mice with accumulation of aberrantly activated astrocytes
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Incubation:

Article Title: Innate immune adaptor TRIF deficiency accelerates disease progression of ALS mice with accumulation of aberrantly activated astrocytes
Article Snippet: Flow cytometry analysis of spinal cord immune cells The spinal cord, dissected from mice transcardially perfused with PBS, was minced into 1 mm3 pieces in solution containing 1 mg/mL or 0.1 mg/mL collagenase IV (Worthington Biochemical Corporation, USA) plus 0.4 mg/mL DNase I (Roche, Switzerland), and incubated at 37 °C for 15 min. For isolation of immune cells and microglia, cells were resuspended in 37% Percoll (GE Healthcare, USA) and centrifuged at 780 × g for 20 min. After centrifugation, myelin debris was removed and the cell pellet was collected. .. Cells were incubated with anti-CD16/CD32 antibodies (eBioscience, USA) for blocking Fc receptors, and then stained with combinations of the following antibodies: anti-CD45-PE-Cy7, anti-CD45-APC-Cy7, anti-CD4-PE, anti-CD8a-FITC, anti-CD8a-APC-Cy7, anti-NK1.1-PerCP-Cy5.5, anti-NK1.1-PE, anti-CD11c-PE, anti-CD11b-APC-Cy7, anti-CD11b-PerCP-Cy5.5 (BD Biosciences, USA), anti-CD4-APC, anti-CD3e-PerCP-Cy5.5, anti-CD86-APC, anti-CD25-PE (eBioscience, USA), anti-CD11c-APC, anti-I-A/I-E (MHC class II)-FITC, anti-CD4-PE-Cy7, anti-CD4-APC, anti-CD68-PE, anti-H-2Kb /H-2Db (MHC class I)-PE, anti-CD206-PE, anti-CD178 (FasL)-PE, and Armenian Hamster IgG Isotype control-PE (BioLegend, USA). .. Flow cytometry was performed by using FACS Aria and FACS Verse flow cytometers (BD Biosciences, USA) and the data was further analyzed using FlowJo Software (FlowJo, LLC, USA).

Blocking Assay:

Article Title: Innate immune adaptor TRIF deficiency accelerates disease progression of ALS mice with accumulation of aberrantly activated astrocytes
Article Snippet: Flow cytometry analysis of spinal cord immune cells The spinal cord, dissected from mice transcardially perfused with PBS, was minced into 1 mm3 pieces in solution containing 1 mg/mL or 0.1 mg/mL collagenase IV (Worthington Biochemical Corporation, USA) plus 0.4 mg/mL DNase I (Roche, Switzerland), and incubated at 37 °C for 15 min. For isolation of immune cells and microglia, cells were resuspended in 37% Percoll (GE Healthcare, USA) and centrifuged at 780 × g for 20 min. After centrifugation, myelin debris was removed and the cell pellet was collected. .. Cells were incubated with anti-CD16/CD32 antibodies (eBioscience, USA) for blocking Fc receptors, and then stained with combinations of the following antibodies: anti-CD45-PE-Cy7, anti-CD45-APC-Cy7, anti-CD4-PE, anti-CD8a-FITC, anti-CD8a-APC-Cy7, anti-NK1.1-PerCP-Cy5.5, anti-NK1.1-PE, anti-CD11c-PE, anti-CD11b-APC-Cy7, anti-CD11b-PerCP-Cy5.5 (BD Biosciences, USA), anti-CD4-APC, anti-CD3e-PerCP-Cy5.5, anti-CD86-APC, anti-CD25-PE (eBioscience, USA), anti-CD11c-APC, anti-I-A/I-E (MHC class II)-FITC, anti-CD4-PE-Cy7, anti-CD4-APC, anti-CD68-PE, anti-H-2Kb /H-2Db (MHC class I)-PE, anti-CD206-PE, anti-CD178 (FasL)-PE, and Armenian Hamster IgG Isotype control-PE (BioLegend, USA). .. Flow cytometry was performed by using FACS Aria and FACS Verse flow cytometers (BD Biosciences, USA) and the data was further analyzed using FlowJo Software (FlowJo, LLC, USA).

IA:

Article Title: The antigenic determinant that defines thymic nurse cells is expressed by thymic epithelial progenitor cells
Article Snippet: Images were acquired using the Zeiss LSM510 Confocal Microscope. .. Primary antibodies used were as follows: rat anti-mouse pH91 monoclonal antibody (IgG2a), K8 TROMA-I (IgG2a) (Developmental Studies Hybridoma Bank, Iowa City, IA), chicken anti-mouse K8 polyclonal antibody (IgY) (Abcam, Cambridge, MA), goat anti-rabbit K5 polyclonal antibody PRB-160B (IgG) (Covance, Princeton, NJ), rabbit anti-goat Δ Np63 (N-16): sc-8609 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Foxn1 polyclonal antibody (IgG) H-270 (Santa Cruz Biotechnology), antibody FITC-conjugated anti-mouse MHC class II (Miltenyi Biotech), biotinylated anti-mouse αβ TCR (BD Pharmingen, San Jose, CA), APC-conjugated CD4 (BD Pharmingen), PE-conjugated CD8 (BD Pharmingen), FITC-conjugated Thy 1.2 (BD Pharmingen), FITC-conjugated rat IgG2a isotype control (BD Pharmingen), and TRITC-conjugated rabbit IgG2a isotype control (BD Pharmingen). .. Secondary antibodies used are as follows: FITC-conjugated mouse anti-rat IgG2a (BD Pharmingen), APC-conjugated donkey anti-chicken IgY (Jackson ImmunoResearch Laboratories, West Grove, PA), TRITC-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories), APC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories), TRITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories), TRITC-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories), TRITC-conjugated rabbit anti-goat IgG (Jackson ImmunoResearch Laboratories), and TRITC-conjugated streptavidin (BD Pharmingen).

FACS:

Article Title: VEGFR1/ CXCR4 positive progenitor cells modulate local inflammation and augment tissue perfusion by a SDF-1 dependent mechanism
Article Snippet: To enable identification of transplanted cells, MDPCs were transduced with a retrovirus carrying the LacZ plasmid pCL-MFG-LacZ (Imgenex, San Diego, California). .. MDPCs were collected with trypsin/EDTA and washed with FACS wash (PBS, 2mM EDTA and 0.5% BSA) and stained with the following labeled antibodies: anti-CD90 PE (clone PX-7), anti-CD44 FITC (clone OX-49), anti-MHC Class I PE (clone OX-18), anti-MHC Class II PE (clone OX-6), anti-CD45 PE (clone OX-1), and PE and FITC isotype controls (all BD Biosciences, San Diego, California). .. The following unlabeled antibodies were also used: anti-CXCR4 (LS-C6307; LifeSpan BioSciences, Seattle, Washington), anti-VEGFR1 (ab2350; Abcam, Hartford, Connecticut, USA), anti-CD73 (clone 5F/B9; BD Biosciences), and corresponding unlabeled isotype controls.

Labeling:

Article Title: VEGFR1/ CXCR4 positive progenitor cells modulate local inflammation and augment tissue perfusion by a SDF-1 dependent mechanism
Article Snippet: To enable identification of transplanted cells, MDPCs were transduced with a retrovirus carrying the LacZ plasmid pCL-MFG-LacZ (Imgenex, San Diego, California). .. MDPCs were collected with trypsin/EDTA and washed with FACS wash (PBS, 2mM EDTA and 0.5% BSA) and stained with the following labeled antibodies: anti-CD90 PE (clone PX-7), anti-CD44 FITC (clone OX-49), anti-MHC Class I PE (clone OX-18), anti-MHC Class II PE (clone OX-6), anti-CD45 PE (clone OX-1), and PE and FITC isotype controls (all BD Biosciences, San Diego, California). .. The following unlabeled antibodies were also used: anti-CXCR4 (LS-C6307; LifeSpan BioSciences, Seattle, Washington), anti-VEGFR1 (ab2350; Abcam, Hartford, Connecticut, USA), anti-CD73 (clone 5F/B9; BD Biosciences), and corresponding unlabeled isotype controls.

Recombinant:

Article Title: Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans
Article Snippet: CFSE (Sigma-Aldrich) and LE540 (Wako Chemicals USA, Inc.) were dissolved in DMSO. .. Cell staining reagents The following reagents were used to stain murine cells: unconjugated or directly conjugated anti-CD103 (M290), anti-α4β7 (DATK32), anti-CD62L (MEL-14), anti-MHC class II (2G9), anti-CD11c (N418), and rat IgG2a and IgG2b isotype controls (BD Biosciences); perCP-Cy5.5, Alexa 700–conjugated anti-CD11c (N418), and Pacific blue–conjugated anti–MHC Class II (M5/114.15.2; BioLegend); anti-CCR7 (4B12; eBiosciences); recombinant mouse E-selectin human Fc chimera (R & D Systems); anti-FcRII/III (2.4G2), anti-CD40 (FGK45), anti-CD86 (GL1), and anti-CCR9 antibody (7E7, ( ); purified from hybridomas); Cy5-labeled donkey anti–human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories); biotinylated mouse anti–rat IgG2a (RG7/1.30), biotinylated mouse anti–rat IgG2b (G15-337), PE-labeled Ki67, streptavidin-conjugated PE, PECy7, PerCP Cy5.5, and the BrdU FITC flow kit (BD Biosciences); streptavidin–alexa 488 (Invitrogen); and DAPI (Sigma-Aldrich). .. The following reagents were used to stain human DCs: APC-conjugated anti-CD40 (5C3), APC-Cy7–conjugated anti–HLA-DR (L243), PE-Cy5–conjugated anti-β7 (FIB504), and APC-Cy7–conjugated anti-CD8 (SK7; BD Biosciences); PE-Cy7–conjugated anti-CD11c (3.9), biotinylated or PE-Cy5–conjugated anti-CD83 (HB15e), PE-conjugated anti-CD103 (BLy7), APC/PE/PE-Cy5– or biotin-conjugated IgG1 isotype controls, and FITC-conjugated anti-CD3 (UCHT1), -CD14 (61D3), -CD16 (CB16), -CD19 (HIB19), and -CD56 (MEM188; eBiosciences); PE-Cy7–conjugated anti-CD3 (UCHT1; Beckman Coulter); APC-conjugated anti-CCR9 (R & D Systems); PE-conjugated anti-CD8 (DK2) and FITC/PE-conjugated anti-CD103 (Ber–ACT8; Dako); FITC/Cy7-conjugated anti-CD11c (BU15) and FITC/Cy5-conjugated anti–HLA-DR (YE2/36HLK; AbD Serotec); and mouse anti–human α4β7 (ACT-1; provided by M. Briskin, Millennium Pharmaceuticals, Inc.).

Purification:

Article Title: Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans
Article Snippet: CFSE (Sigma-Aldrich) and LE540 (Wako Chemicals USA, Inc.) were dissolved in DMSO. .. Cell staining reagents The following reagents were used to stain murine cells: unconjugated or directly conjugated anti-CD103 (M290), anti-α4β7 (DATK32), anti-CD62L (MEL-14), anti-MHC class II (2G9), anti-CD11c (N418), and rat IgG2a and IgG2b isotype controls (BD Biosciences); perCP-Cy5.5, Alexa 700–conjugated anti-CD11c (N418), and Pacific blue–conjugated anti–MHC Class II (M5/114.15.2; BioLegend); anti-CCR7 (4B12; eBiosciences); recombinant mouse E-selectin human Fc chimera (R & D Systems); anti-FcRII/III (2.4G2), anti-CD40 (FGK45), anti-CD86 (GL1), and anti-CCR9 antibody (7E7, ( ); purified from hybridomas); Cy5-labeled donkey anti–human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories); biotinylated mouse anti–rat IgG2a (RG7/1.30), biotinylated mouse anti–rat IgG2b (G15-337), PE-labeled Ki67, streptavidin-conjugated PE, PECy7, PerCP Cy5.5, and the BrdU FITC flow kit (BD Biosciences); streptavidin–alexa 488 (Invitrogen); and DAPI (Sigma-Aldrich). .. The following reagents were used to stain human DCs: APC-conjugated anti-CD40 (5C3), APC-Cy7–conjugated anti–HLA-DR (L243), PE-Cy5–conjugated anti-β7 (FIB504), and APC-Cy7–conjugated anti-CD8 (SK7; BD Biosciences); PE-Cy7–conjugated anti-CD11c (3.9), biotinylated or PE-Cy5–conjugated anti-CD83 (HB15e), PE-conjugated anti-CD103 (BLy7), APC/PE/PE-Cy5– or biotin-conjugated IgG1 isotype controls, and FITC-conjugated anti-CD3 (UCHT1), -CD14 (61D3), -CD16 (CB16), -CD19 (HIB19), and -CD56 (MEM188; eBiosciences); PE-Cy7–conjugated anti-CD3 (UCHT1; Beckman Coulter); APC-conjugated anti-CCR9 (R & D Systems); PE-conjugated anti-CD8 (DK2) and FITC/PE-conjugated anti-CD103 (Ber–ACT8; Dako); FITC/Cy7-conjugated anti-CD11c (BU15) and FITC/Cy5-conjugated anti–HLA-DR (YE2/36HLK; AbD Serotec); and mouse anti–human α4β7 (ACT-1; provided by M. Briskin, Millennium Pharmaceuticals, Inc.).

Flow Cytometry:

Article Title: Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans
Article Snippet: CFSE (Sigma-Aldrich) and LE540 (Wako Chemicals USA, Inc.) were dissolved in DMSO. .. Cell staining reagents The following reagents were used to stain murine cells: unconjugated or directly conjugated anti-CD103 (M290), anti-α4β7 (DATK32), anti-CD62L (MEL-14), anti-MHC class II (2G9), anti-CD11c (N418), and rat IgG2a and IgG2b isotype controls (BD Biosciences); perCP-Cy5.5, Alexa 700–conjugated anti-CD11c (N418), and Pacific blue–conjugated anti–MHC Class II (M5/114.15.2; BioLegend); anti-CCR7 (4B12; eBiosciences); recombinant mouse E-selectin human Fc chimera (R & D Systems); anti-FcRII/III (2.4G2), anti-CD40 (FGK45), anti-CD86 (GL1), and anti-CCR9 antibody (7E7, ( ); purified from hybridomas); Cy5-labeled donkey anti–human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories); biotinylated mouse anti–rat IgG2a (RG7/1.30), biotinylated mouse anti–rat IgG2b (G15-337), PE-labeled Ki67, streptavidin-conjugated PE, PECy7, PerCP Cy5.5, and the BrdU FITC flow kit (BD Biosciences); streptavidin–alexa 488 (Invitrogen); and DAPI (Sigma-Aldrich). .. The following reagents were used to stain human DCs: APC-conjugated anti-CD40 (5C3), APC-Cy7–conjugated anti–HLA-DR (L243), PE-Cy5–conjugated anti-β7 (FIB504), and APC-Cy7–conjugated anti-CD8 (SK7; BD Biosciences); PE-Cy7–conjugated anti-CD11c (3.9), biotinylated or PE-Cy5–conjugated anti-CD83 (HB15e), PE-conjugated anti-CD103 (BLy7), APC/PE/PE-Cy5– or biotin-conjugated IgG1 isotype controls, and FITC-conjugated anti-CD3 (UCHT1), -CD14 (61D3), -CD16 (CB16), -CD19 (HIB19), and -CD56 (MEM188; eBiosciences); PE-Cy7–conjugated anti-CD3 (UCHT1; Beckman Coulter); APC-conjugated anti-CCR9 (R & D Systems); PE-conjugated anti-CD8 (DK2) and FITC/PE-conjugated anti-CD103 (Ber–ACT8; Dako); FITC/Cy7-conjugated anti-CD11c (BU15) and FITC/Cy5-conjugated anti–HLA-DR (YE2/36HLK; AbD Serotec); and mouse anti–human α4β7 (ACT-1; provided by M. Briskin, Millennium Pharmaceuticals, Inc.).

Activation Assay:

Article Title: Dynamic cross-regulation of antigen-specific effector and regulatory T cell subpopulations and microglia in brain autoimmunity
Article Snippet: After washing, cells were stained with 7-AAD, (BD Pharmingen) and antibodies against CD4 (BD Pharmingen), CD62L (BD Pharmingen), CD25 (BD Pharmingen), CD69 (BD Pharmingen), and CD45 (BD Pharmingen). .. For microglia activation, cell were stained with anti-MHC class II (IAb ) (Abcam), CD11b (BD Pharmingen) and CD45 (BD Pharmingen). .. B-cell staining was performed using anti CD45R/B220 (BD Pharmingen) and anti-CD21 (BD Pharmingen) antibodies.

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  • 86
    Becton Dickinson anti mhc class ii
    Turnover of SI-LP and MLN <t>CD103</t> + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs <t>(MHC</t> class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).
    Anti Mhc Class Ii, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Migrating CFSE + cDC transport viral RNA to the lung-draining LN and present antigens by <t>MHC-I</t> and MHC-II molecules. (A) MLN cDC were harvested 72 h post-RSV infection and sorted into four populations based on <t>CD103</t> expression and CFSE labeling.
    Anti Mhc Ii, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti hla dr sorbent
    Phenotypic analysis of IFN-DC generated in vitro with different stimuli. Immature IFN-DC were cultured with different stimuli for 24 h followed by flow cytometry analysis of surface molecules CD14, CD83, <t>HLA-DR,</t> <t>CD86,</t> OX40L. Figures show flow cytometry histograms representing the expression of studied markers (bold-line histograms) and matched isotype controls (gray-filled histograms).
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    Becton Dickinson hla dr
    LRRC32 + CD4 + CD25 hi FoxP3 T regs appear to be more potent suppressors than LRRC32 - CD4 + CD25 hi FoxP3 and exhibit decreased <t>CD62L</t> upon activation . a) Expression of LRRC32 and LAP in CD4+ T cells rested overnight (top panel) or stimulated with plate bound anti-CD3 and soluble anti-CD28 (bottom panel). T regs were selected from the top 5% CD25-expressing and FoxP3 + populations, as previously described. Confirmation of activation by expression of the surface markers CD40L and CD69 are also shown (top of each panel). b) The expression patterns of various T reg and activation surface markers (CD62L, CD69, GITR, CTLA4, CD45RO, and <t>HLA-DR)</t> in FoxP3 + and LRRC32 + -gated populations of CD25 hi cells were studied using flow cytometry. Stimulated CD4 + FoxP3 + CD25 hi T regs (top panel) unstimulated CD4 + FoxP3 + CD25 hi T regs (bottom panel). c) Composite summary of phenotypic analysis of unstimulated LRRC32 - CD4 + CD25 hi FoxP3 + T regs and stimulated LRRC32 + and LRRC32 - CD4 + CD25 hi FoxP3 + T regs . Black bars = unstimulated LRRC32 - T regs . Dark grey bars = stimulated LRRC32 - T regs . Light grey bars = stimulated LRRC32 + T regs . Data are expressed as the mean ± SEM from 3 individuals. Heteroscedastic variances and an independent t-test comparing stimulated LRRC32 + and LRRC32 - subsets were used for calculations of the p values which are reported along the x-axis, below each surface marker (*). d) CD25 hi cells were sorted and activated overnight using anti-CD3-coated plates and soluble anti-CD28 (1 microgram/ml). Cells were then resorted based upon LRRC32 expression. The suppressive capacities of these LRRC32 + and LRRC32 - T regs were subsequently tested in a mixed lymphocyte reaction utilizing syngeneic effectors (T eff , 20,000/well) and allogenic antigen presenting cells (50,000/well). T reg :T eff ratios are depicted above. Data summarize 3 independent experiments. Results are expressed as the mean ± SEM. p = 0.0001 and R 2 = 0.7244. Absolute proliferation values for the 3 experiments were as follow: T effs alone: average of 31094 cpm to average of 47483 cpm (at least 6 replicates per assay), background: average of 24 cpm to 35 cpm (at least 6 replicates per assay); T reg :T eff ratio of 1:1: 89 cpm to 346 cpm. When titrating T regs vs. T effs , 3 replicates were performed at each titration for the LRRC32 + and LRRC32 - T regs except for in one assay set in which there was limited number of LRRC32 + T regs . In this case, only one replicate was performed at the 1:1 and 1:2 titrations, and two replicates were performed for the other titrations (0:1, 1:4, 1:8, and 1:16). We performed 3 replicates for each titration utilizing the LRRC32 - T regs .
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    Turnover of SI-LP and MLN CD103 + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs (MHC class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).

    Journal: The Journal of Experimental Medicine

    Article Title: Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

    doi: 10.1084/jem.20080414

    Figure Lengend Snippet: Turnover of SI-LP and MLN CD103 + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs (MHC class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).

    Article Snippet: Cell staining reagents The following reagents were used to stain murine cells: unconjugated or directly conjugated anti-CD103 (M290), anti-α4β7 (DATK32), anti-CD62L (MEL-14), anti-MHC class II (2G9), anti-CD11c (N418), and rat IgG2a and IgG2b isotype controls (BD Biosciences); perCP-Cy5.5, Alexa 700–conjugated anti-CD11c (N418), and Pacific blue–conjugated anti–MHC Class II (M5/114.15.2; BioLegend); anti-CCR7 (4B12; eBiosciences); recombinant mouse E-selectin human Fc chimera (R & D Systems); anti-FcRII/III (2.4G2), anti-CD40 (FGK45), anti-CD86 (GL1), and anti-CCR9 antibody (7E7, ( ); purified from hybridomas); Cy5-labeled donkey anti–human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories); biotinylated mouse anti–rat IgG2a (RG7/1.30), biotinylated mouse anti–rat IgG2b (G15-337), PE-labeled Ki67, streptavidin-conjugated PE, PECy7, PerCP Cy5.5, and the BrdU FITC flow kit (BD Biosciences); streptavidin–alexa 488 (Invitrogen); and DAPI (Sigma-Aldrich).

    Techniques: Mouse Assay, Injection, Flow Cytometry, Cytometry, Staining, Immunohistochemistry, Derivative Assay

    Localization of CD103 + and CD103 − MHC class II + CD11c + cells within the small intestinal mucosa. (A and B) Expression of CD103 by DCs in small intestinal villus and SILT. (A) Images show overlays of CD103 (red) in combination with CD11c (blue) and MHC class II (green), with insets showing DAPI staining. Arrows point toward MHC class II hi CD11c + DCs coexpressing CD103. Bars, 50 μm. (B) The graph is percentage of MHC class II hi CD11c + cells in SI villus or dome region of SILT that express CD103. Results are mean and SD ( n = 5 mice per group). ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

    doi: 10.1084/jem.20080414

    Figure Lengend Snippet: Localization of CD103 + and CD103 − MHC class II + CD11c + cells within the small intestinal mucosa. (A and B) Expression of CD103 by DCs in small intestinal villus and SILT. (A) Images show overlays of CD103 (red) in combination with CD11c (blue) and MHC class II (green), with insets showing DAPI staining. Arrows point toward MHC class II hi CD11c + DCs coexpressing CD103. Bars, 50 μm. (B) The graph is percentage of MHC class II hi CD11c + cells in SI villus or dome region of SILT that express CD103. Results are mean and SD ( n = 5 mice per group). ***, P

    Article Snippet: Cell staining reagents The following reagents were used to stain murine cells: unconjugated or directly conjugated anti-CD103 (M290), anti-α4β7 (DATK32), anti-CD62L (MEL-14), anti-MHC class II (2G9), anti-CD11c (N418), and rat IgG2a and IgG2b isotype controls (BD Biosciences); perCP-Cy5.5, Alexa 700–conjugated anti-CD11c (N418), and Pacific blue–conjugated anti–MHC Class II (M5/114.15.2; BioLegend); anti-CCR7 (4B12; eBiosciences); recombinant mouse E-selectin human Fc chimera (R & D Systems); anti-FcRII/III (2.4G2), anti-CD40 (FGK45), anti-CD86 (GL1), and anti-CCR9 antibody (7E7, ( ); purified from hybridomas); Cy5-labeled donkey anti–human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories); biotinylated mouse anti–rat IgG2a (RG7/1.30), biotinylated mouse anti–rat IgG2b (G15-337), PE-labeled Ki67, streptavidin-conjugated PE, PECy7, PerCP Cy5.5, and the BrdU FITC flow kit (BD Biosciences); streptavidin–alexa 488 (Invitrogen); and DAPI (Sigma-Aldrich).

    Techniques: Expressing, Staining, Mouse Assay

    Migrating CFSE + cDC transport viral RNA to the lung-draining LN and present antigens by MHC-I and MHC-II molecules. (A) MLN cDC were harvested 72 h post-RSV infection and sorted into four populations based on CD103 expression and CFSE labeling.

    Journal: Journal of Virology

    Article Title: Respiratory Syncytial Virus-Induced Activation and Migration of Respiratory Dendritic Cells and Subsequent Antigen Presentation in the Lung-Draining Lymph Node ▿

    doi: 10.1128/JVI.00452-09

    Figure Lengend Snippet: Migrating CFSE + cDC transport viral RNA to the lung-draining LN and present antigens by MHC-I and MHC-II molecules. (A) MLN cDC were harvested 72 h post-RSV infection and sorted into four populations based on CD103 expression and CFSE labeling.

    Article Snippet: The cells were stained in PBS containing 2% fetal calf serum, 2 mM EDTA, and 0.02% NaN3 with the following monoclonal antibodies: anti-CD4 (L3T4), anti-CD8α (clone 53-6.7), anti-CD11b (clone M1/70), anti-CD11c (clone HL3), anti-CD19 (clone 1D3), anti-CD40 (clone 3/23), anti-CD45R (B220, clone RA3-6B2), anti-CD80 (clone 16-10A1), anti-CD86 (clone GL1), anti-CD103 (clone M290), and anti-MHC-II (I-Ab /I-Eb ) (clone M5/114.15.2), obtained from BD Biosciences (San Diego, CA), and anti-mPDCA-1 (clone JF05-1C2.4.1), obtained from Miltenyi Biotec (Germany).

    Techniques: Infection, Expressing, Labeling

    Phenotypic analysis of IFN-DC generated in vitro with different stimuli. Immature IFN-DC were cultured with different stimuli for 24 h followed by flow cytometry analysis of surface molecules CD14, CD83, HLA-DR, CD86, OX40L. Figures show flow cytometry histograms representing the expression of studied markers (bold-line histograms) and matched isotype controls (gray-filled histograms).

    Journal: Vavilov Journal of Genetics and Breeding

    Article Title: Phosphate-modif ied CpG oligonucleotides induce in vitro maturation of human myeloid dendritic cells

    doi: 10.18699/VJ20.659

    Figure Lengend Snippet: Phenotypic analysis of IFN-DC generated in vitro with different stimuli. Immature IFN-DC were cultured with different stimuli for 24 h followed by flow cytometry analysis of surface molecules CD14, CD83, HLA-DR, CD86, OX40L. Figures show flow cytometry histograms representing the expression of studied markers (bold-line histograms) and matched isotype controls (gray-filled histograms).

    Article Snippet: To assess proliferation, cells were incubated for 4 days, followed by pulse-labelling with 1.0 μCi/well of [3H] thymidine for the last 18 h. To perform immunophenotyping of IFN-DC, cells were stained with phycoerythrin (PE)-labelled anti-СD14 (Sorbent, Moscow) or anti-OX40L (anti-CD252, BioLegend, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-labelled anti- СD83, anti-CD86 (BD PharMingen) and anti-HLA-DR (Sorbent), and analysed by flow cytometry (FACSCalibur, Becton Dickinson).

    Techniques: Generated, In Vitro, Cell Culture, Flow Cytometry, Expressing

    LRRC32 + CD4 + CD25 hi FoxP3 T regs appear to be more potent suppressors than LRRC32 - CD4 + CD25 hi FoxP3 and exhibit decreased CD62L upon activation . a) Expression of LRRC32 and LAP in CD4+ T cells rested overnight (top panel) or stimulated with plate bound anti-CD3 and soluble anti-CD28 (bottom panel). T regs were selected from the top 5% CD25-expressing and FoxP3 + populations, as previously described. Confirmation of activation by expression of the surface markers CD40L and CD69 are also shown (top of each panel). b) The expression patterns of various T reg and activation surface markers (CD62L, CD69, GITR, CTLA4, CD45RO, and HLA-DR) in FoxP3 + and LRRC32 + -gated populations of CD25 hi cells were studied using flow cytometry. Stimulated CD4 + FoxP3 + CD25 hi T regs (top panel) unstimulated CD4 + FoxP3 + CD25 hi T regs (bottom panel). c) Composite summary of phenotypic analysis of unstimulated LRRC32 - CD4 + CD25 hi FoxP3 + T regs and stimulated LRRC32 + and LRRC32 - CD4 + CD25 hi FoxP3 + T regs . Black bars = unstimulated LRRC32 - T regs . Dark grey bars = stimulated LRRC32 - T regs . Light grey bars = stimulated LRRC32 + T regs . Data are expressed as the mean ± SEM from 3 individuals. Heteroscedastic variances and an independent t-test comparing stimulated LRRC32 + and LRRC32 - subsets were used for calculations of the p values which are reported along the x-axis, below each surface marker (*). d) CD25 hi cells were sorted and activated overnight using anti-CD3-coated plates and soluble anti-CD28 (1 microgram/ml). Cells were then resorted based upon LRRC32 expression. The suppressive capacities of these LRRC32 + and LRRC32 - T regs were subsequently tested in a mixed lymphocyte reaction utilizing syngeneic effectors (T eff , 20,000/well) and allogenic antigen presenting cells (50,000/well). T reg :T eff ratios are depicted above. Data summarize 3 independent experiments. Results are expressed as the mean ± SEM. p = 0.0001 and R 2 = 0.7244. Absolute proliferation values for the 3 experiments were as follow: T effs alone: average of 31094 cpm to average of 47483 cpm (at least 6 replicates per assay), background: average of 24 cpm to 35 cpm (at least 6 replicates per assay); T reg :T eff ratio of 1:1: 89 cpm to 346 cpm. When titrating T regs vs. T effs , 3 replicates were performed at each titration for the LRRC32 + and LRRC32 - T regs except for in one assay set in which there was limited number of LRRC32 + T regs . In this case, only one replicate was performed at the 1:1 and 1:2 titrations, and two replicates were performed for the other titrations (0:1, 1:4, 1:8, and 1:16). We performed 3 replicates for each titration utilizing the LRRC32 - T regs .

    Journal: BMC Biochemistry

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

    doi: 10.1186/1471-2091-12-27

    Figure Lengend Snippet: LRRC32 + CD4 + CD25 hi FoxP3 T regs appear to be more potent suppressors than LRRC32 - CD4 + CD25 hi FoxP3 and exhibit decreased CD62L upon activation . a) Expression of LRRC32 and LAP in CD4+ T cells rested overnight (top panel) or stimulated with plate bound anti-CD3 and soluble anti-CD28 (bottom panel). T regs were selected from the top 5% CD25-expressing and FoxP3 + populations, as previously described. Confirmation of activation by expression of the surface markers CD40L and CD69 are also shown (top of each panel). b) The expression patterns of various T reg and activation surface markers (CD62L, CD69, GITR, CTLA4, CD45RO, and HLA-DR) in FoxP3 + and LRRC32 + -gated populations of CD25 hi cells were studied using flow cytometry. Stimulated CD4 + FoxP3 + CD25 hi T regs (top panel) unstimulated CD4 + FoxP3 + CD25 hi T regs (bottom panel). c) Composite summary of phenotypic analysis of unstimulated LRRC32 - CD4 + CD25 hi FoxP3 + T regs and stimulated LRRC32 + and LRRC32 - CD4 + CD25 hi FoxP3 + T regs . Black bars = unstimulated LRRC32 - T regs . Dark grey bars = stimulated LRRC32 - T regs . Light grey bars = stimulated LRRC32 + T regs . Data are expressed as the mean ± SEM from 3 individuals. Heteroscedastic variances and an independent t-test comparing stimulated LRRC32 + and LRRC32 - subsets were used for calculations of the p values which are reported along the x-axis, below each surface marker (*). d) CD25 hi cells were sorted and activated overnight using anti-CD3-coated plates and soluble anti-CD28 (1 microgram/ml). Cells were then resorted based upon LRRC32 expression. The suppressive capacities of these LRRC32 + and LRRC32 - T regs were subsequently tested in a mixed lymphocyte reaction utilizing syngeneic effectors (T eff , 20,000/well) and allogenic antigen presenting cells (50,000/well). T reg :T eff ratios are depicted above. Data summarize 3 independent experiments. Results are expressed as the mean ± SEM. p = 0.0001 and R 2 = 0.7244. Absolute proliferation values for the 3 experiments were as follow: T effs alone: average of 31094 cpm to average of 47483 cpm (at least 6 replicates per assay), background: average of 24 cpm to 35 cpm (at least 6 replicates per assay); T reg :T eff ratio of 1:1: 89 cpm to 346 cpm. When titrating T regs vs. T effs , 3 replicates were performed at each titration for the LRRC32 + and LRRC32 - T regs except for in one assay set in which there was limited number of LRRC32 + T regs . In this case, only one replicate was performed at the 1:1 and 1:2 titrations, and two replicates were performed for the other titrations (0:1, 1:4, 1:8, and 1:16). We performed 3 replicates for each titration utilizing the LRRC32 - T regs .

    Article Snippet: Cells were subsequently stained using antibodies to CD25 (clone 2A3, Becton Dickenson), LRRC32 (Axxora), FoxP3 (eBioscience), and a panel of antibodies specific for CD69 (Becton Dickinson), CD62L (Becton Dickinson), GITR (R & D Biosystems), CTLA4 (Becton Dickinson), HLA-DR (Becton Dickinson), and CD45RO (Becton Dickinson), per manufacturer's protocol for staining cells for flow cytometry (eBioscience).

    Techniques: Activation Assay, Expressing, Flow Cytometry, Marker, Titration