Structured Review

Agilent technologies mouse anti human cd45
Effects of OXi4503 and bevacizumab on subcutaneous leukemic chloromas . NOD/scid/IL2Rγ −/− (NOG) mice were subcutaneously inoculated with KG-1 human acute myelogenous leukemia (AML) cells. After chloromas were palpable mice were treated with intraperitoneal injections of bevacizumab, OXi4503, combination (OXi+Bev), or controls. Leukemia growth was measured every other day. (A) OXi4503 alone decreased leukemia growth in comparison to controls. Combination Oxi4503+Bev treatment resulted in regression of cancer size. Bevacizumab alone had no effect on tumor growth. (B) Comprehensive staining of chloromas was performed for: H E (scale bar: 200 μm), TUNEL/MECA-32 (scale bar: 100 μm), <t>(TUNEL/CD45</t> (scale bar: 50 μm), and CD45/VEGF-A (scale bar: 100 μm). Sections showed that OXi4503 monotherapy led to chloromas with central cores made up mainly of nonvascularized, TUNEL + apoptotic cells and viable rims (outlined by dotted lines) containing MECA-32 + blood vessels at the periphery of leukemias. VEGF-A expression was also observed in viable rims. Combination therapy eliminated the viable rim resulting in widespread apoptosis and a lack of intact blood vessels throughout the tumor mass. Bevacizumab-treated tumors showed no difference in staining compared with controls. (C) Quantification of microvessels based on MECA-32 + blood vessels revealed decreased density within leukemic cores of mice treated with OXi4503 and combination therapy in comparison to bulk control tumors. Blood vessels within viable leukemia rims are increased after OXi4503 treatment but significantly decreased with additional bevacizumab treatment. Values represent mean ± SEM. * P
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1) Product Images from "Leukemia regression by vascular disruption and antiangiogenic therapy"

Article Title: Leukemia regression by vascular disruption and antiangiogenic therapy

Journal: Blood

doi: 10.1182/blood-2009-06-230474

Effects of OXi4503 and bevacizumab on subcutaneous leukemic chloromas . NOD/scid/IL2Rγ −/− (NOG) mice were subcutaneously inoculated with KG-1 human acute myelogenous leukemia (AML) cells. After chloromas were palpable mice were treated with intraperitoneal injections of bevacizumab, OXi4503, combination (OXi+Bev), or controls. Leukemia growth was measured every other day. (A) OXi4503 alone decreased leukemia growth in comparison to controls. Combination Oxi4503+Bev treatment resulted in regression of cancer size. Bevacizumab alone had no effect on tumor growth. (B) Comprehensive staining of chloromas was performed for: H E (scale bar: 200 μm), TUNEL/MECA-32 (scale bar: 100 μm), (TUNEL/CD45 (scale bar: 50 μm), and CD45/VEGF-A (scale bar: 100 μm). Sections showed that OXi4503 monotherapy led to chloromas with central cores made up mainly of nonvascularized, TUNEL + apoptotic cells and viable rims (outlined by dotted lines) containing MECA-32 + blood vessels at the periphery of leukemias. VEGF-A expression was also observed in viable rims. Combination therapy eliminated the viable rim resulting in widespread apoptosis and a lack of intact blood vessels throughout the tumor mass. Bevacizumab-treated tumors showed no difference in staining compared with controls. (C) Quantification of microvessels based on MECA-32 + blood vessels revealed decreased density within leukemic cores of mice treated with OXi4503 and combination therapy in comparison to bulk control tumors. Blood vessels within viable leukemia rims are increased after OXi4503 treatment but significantly decreased with additional bevacizumab treatment. Values represent mean ± SEM. * P
Figure Legend Snippet: Effects of OXi4503 and bevacizumab on subcutaneous leukemic chloromas . NOD/scid/IL2Rγ −/− (NOG) mice were subcutaneously inoculated with KG-1 human acute myelogenous leukemia (AML) cells. After chloromas were palpable mice were treated with intraperitoneal injections of bevacizumab, OXi4503, combination (OXi+Bev), or controls. Leukemia growth was measured every other day. (A) OXi4503 alone decreased leukemia growth in comparison to controls. Combination Oxi4503+Bev treatment resulted in regression of cancer size. Bevacizumab alone had no effect on tumor growth. (B) Comprehensive staining of chloromas was performed for: H E (scale bar: 200 μm), TUNEL/MECA-32 (scale bar: 100 μm), (TUNEL/CD45 (scale bar: 50 μm), and CD45/VEGF-A (scale bar: 100 μm). Sections showed that OXi4503 monotherapy led to chloromas with central cores made up mainly of nonvascularized, TUNEL + apoptotic cells and viable rims (outlined by dotted lines) containing MECA-32 + blood vessels at the periphery of leukemias. VEGF-A expression was also observed in viable rims. Combination therapy eliminated the viable rim resulting in widespread apoptosis and a lack of intact blood vessels throughout the tumor mass. Bevacizumab-treated tumors showed no difference in staining compared with controls. (C) Quantification of microvessels based on MECA-32 + blood vessels revealed decreased density within leukemic cores of mice treated with OXi4503 and combination therapy in comparison to bulk control tumors. Blood vessels within viable leukemia rims are increased after OXi4503 treatment but significantly decreased with additional bevacizumab treatment. Values represent mean ± SEM. * P

Techniques Used: Mouse Assay, Staining, TUNEL Assay, Expressing

OXi4503 induced regression of systemic AML in bone marrow . Irradiated NOG mice were transplanted with human AML of differing subtypes (M1/2, M4, and M5) including a primary human AML specimen that harbored a high-risk FLT3 ITD mutation and after verification of leukemia engraftment were randomly assigned to 1 of 4 treatment cohorts. (A) Six weeks after AML transplant, NOG mice were treated with bevacizumab, OXi4503, combination (OXi4503+Bev), or controls. After 2 weeks of treatment, bone marrow showed persistence of AML in control and bevacizumab-treated mice. However, incidences of AML engraftment were significantly decreased with OXi4503 (1/8 positive) and combination treatment (1/11 positive) in comparison to controls. Shown are representative flow cytometric plots showing leukemic engraftment in control and bevacizumab-treated mice and no leukemic engraftment in Oxi4503 and combination-treated mice. (B) Quantification of AML engraftment by flow cytometry showed significantly decreased engraftment in OXi4503 and combination-treated animals versus controls. (C) PCR analysis for FLT3 ITD AML revealed molecular remissions (*) of high-risk FLT3 ITD + AML in 40% of OXi4503 and combination-treated animals. (D-E) TUNEL staining showed no detectable apoptotic response to bevacizumab (D); however, a hypoxia-mediated reaction was observed upon HIF-1α staining (E). CD45 + cells were observed throughout bone marrow sections (scale bar: 100 μm). Values represent mean ± SEM. Gating was established using appropriate isotype controls.
Figure Legend Snippet: OXi4503 induced regression of systemic AML in bone marrow . Irradiated NOG mice were transplanted with human AML of differing subtypes (M1/2, M4, and M5) including a primary human AML specimen that harbored a high-risk FLT3 ITD mutation and after verification of leukemia engraftment were randomly assigned to 1 of 4 treatment cohorts. (A) Six weeks after AML transplant, NOG mice were treated with bevacizumab, OXi4503, combination (OXi4503+Bev), or controls. After 2 weeks of treatment, bone marrow showed persistence of AML in control and bevacizumab-treated mice. However, incidences of AML engraftment were significantly decreased with OXi4503 (1/8 positive) and combination treatment (1/11 positive) in comparison to controls. Shown are representative flow cytometric plots showing leukemic engraftment in control and bevacizumab-treated mice and no leukemic engraftment in Oxi4503 and combination-treated mice. (B) Quantification of AML engraftment by flow cytometry showed significantly decreased engraftment in OXi4503 and combination-treated animals versus controls. (C) PCR analysis for FLT3 ITD AML revealed molecular remissions (*) of high-risk FLT3 ITD + AML in 40% of OXi4503 and combination-treated animals. (D-E) TUNEL staining showed no detectable apoptotic response to bevacizumab (D); however, a hypoxia-mediated reaction was observed upon HIF-1α staining (E). CD45 + cells were observed throughout bone marrow sections (scale bar: 100 μm). Values represent mean ± SEM. Gating was established using appropriate isotype controls.

Techniques Used: Irradiation, Mouse Assay, Mutagenesis, Flow Cytometry, Cytometry, Polymerase Chain Reaction, TUNEL Assay, Staining

2) Product Images from "Human Solid Tumor Xenografts in Immunodeficient Mice Are Vulnerable to Lymphomagenesis Associated with Epstein-Barr Virus"

Article Title: Human Solid Tumor Xenografts in Immunodeficient Mice Are Vulnerable to Lymphomagenesis Associated with Epstein-Barr Virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039294

Expression of leukocyte markers and EBER in non-HCC-like xenografts. (A) Representative section (×200) from a parent HCC sample that gave rise to a non-HCC-like xenograft, demonstrating a typical distribution of CD45 + leukocytes along a portal tract invaded by the tumor, only a small fraction of which are CD20 + B lymphocytes; EBER ISH is negative. (B) Representative sections (×400) from three non-HCC-like xenografts demonstrating that a very high proportion of cells stain positively for human CD45 and human CD20 (brown), consistent with human B lymphocytes; EBER ISH is very strongly positive in the cells in these xenografts (dark blue). (C) Representative multiparameter flow cytometry analysis of freshly isolated cells from a non-HCC-like xenograft demonstrating that a large proportion of tumor cells are human CD45 + leukocytes (left plot), and that the majority of the gated CD45 + population also expresses human CD19 + (right plot), consistent with B lymphocytes.
Figure Legend Snippet: Expression of leukocyte markers and EBER in non-HCC-like xenografts. (A) Representative section (×200) from a parent HCC sample that gave rise to a non-HCC-like xenograft, demonstrating a typical distribution of CD45 + leukocytes along a portal tract invaded by the tumor, only a small fraction of which are CD20 + B lymphocytes; EBER ISH is negative. (B) Representative sections (×400) from three non-HCC-like xenografts demonstrating that a very high proportion of cells stain positively for human CD45 and human CD20 (brown), consistent with human B lymphocytes; EBER ISH is very strongly positive in the cells in these xenografts (dark blue). (C) Representative multiparameter flow cytometry analysis of freshly isolated cells from a non-HCC-like xenograft demonstrating that a large proportion of tumor cells are human CD45 + leukocytes (left plot), and that the majority of the gated CD45 + population also expresses human CD19 + (right plot), consistent with B lymphocytes.

Techniques Used: Expressing, In Situ Hybridization, Staining, Flow Cytometry, Cytometry, Isolation

3) Product Images from "Identification of Hepatic Niche Harboring Human Acute Lymphoblastic Leukemic Cells via the SDF-1/CXCR4 Axis"

Article Title: Identification of Hepatic Niche Harboring Human Acute Lymphoblastic Leukemic Cells via the SDF-1/CXCR4 Axis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027042

Leukemic cells can be engrafted to NOG mice without pre-conditioning. Morphology ( May-Grunwald Giemsa staining) and immunophenotype of leukemic blasts remain stable during serial transplantation in NOG mice. (A) Sequential FACS analyses showing bone marrow engraftment after transplantation. Graphs show percentage of blast cells (hCD45 or hCD19 positive cells) in recipient BM ( n = 3−6 mice per case).Data are shown as means±S.D. (B) H-E staining and anti-hCD45 immunostaining showing increasing number of leukemic cells over time in the humerus of ALL#1 leukemic cell-recipient NOG mice. Scale bar, 10 µm. (C) Morphology and (D) immunophenotype of original patient blast cells (left row) and BM samples derived from murine primary and tertiary transplants of leukemic cells (middle and right rows). Debris (low forward scatter), dead cells (DAPI-positive), and mouse CD45 positive cells were excluded from analysis. Scale bar, 20 µm.
Figure Legend Snippet: Leukemic cells can be engrafted to NOG mice without pre-conditioning. Morphology ( May-Grunwald Giemsa staining) and immunophenotype of leukemic blasts remain stable during serial transplantation in NOG mice. (A) Sequential FACS analyses showing bone marrow engraftment after transplantation. Graphs show percentage of blast cells (hCD45 or hCD19 positive cells) in recipient BM ( n = 3−6 mice per case).Data are shown as means±S.D. (B) H-E staining and anti-hCD45 immunostaining showing increasing number of leukemic cells over time in the humerus of ALL#1 leukemic cell-recipient NOG mice. Scale bar, 10 µm. (C) Morphology and (D) immunophenotype of original patient blast cells (left row) and BM samples derived from murine primary and tertiary transplants of leukemic cells (middle and right rows). Debris (low forward scatter), dead cells (DAPI-positive), and mouse CD45 positive cells were excluded from analysis. Scale bar, 20 µm.

Techniques Used: Mouse Assay, Staining, Transplantation Assay, FACS, Immunostaining, Derivative Assay

4) Product Images from "Natural Killer Group 2D Ligand Depletion Reconstitutes Natural Killer Cell Immunosurveillance of Head and Neck Squamous Cell Carcinoma"

Article Title: Natural Killer Group 2D Ligand Depletion Reconstitutes Natural Killer Cell Immunosurveillance of Head and Neck Squamous Cell Carcinoma

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00387

Low infiltration of lymphocytes in primary head and neck squamous cell carcinoma (HNSCC) . (A) Paraffin sections of primary HNSCC tumors stained for CD3 + /CD8 + , CD20 + or CD56 + lymphocytes, or CD68 + /CD163 + macrophages. Consecutive sections of a representative patient are shown (magnification 200×). (B) Number of infiltrating lymphocytes and macrophages quantified by counting infiltrating cells in High power fields of the tumor slides. Total cell numbers/HPF are plotted as mean ± SEM ( n = 11 patients). (C) Flow cytometric analysis of natural killer (NK) cell subpopulations (CD56 bright /CD16 +/− and CD56 dim /CD16 + ) of patients’ peripheral blood mononuclear cells (PBMCs) and corresponding tumor tissues. PBMCs from healthy donors served as control. NK cell numbers were calculated and plotted as % of CD45 + lymphocytes ( n = 9 patients; n = 4 healthy controls). (D) Corresponding natural killer group 2D (NKG2D) expression levels of NK cell subpopulations plotted as mean fluorescent intensity (MFI) over background ( n = 9 patients). Statistical significance was assessed by one-way analysis of variance. ns, non-significant.
Figure Legend Snippet: Low infiltration of lymphocytes in primary head and neck squamous cell carcinoma (HNSCC) . (A) Paraffin sections of primary HNSCC tumors stained for CD3 + /CD8 + , CD20 + or CD56 + lymphocytes, or CD68 + /CD163 + macrophages. Consecutive sections of a representative patient are shown (magnification 200×). (B) Number of infiltrating lymphocytes and macrophages quantified by counting infiltrating cells in High power fields of the tumor slides. Total cell numbers/HPF are plotted as mean ± SEM ( n = 11 patients). (C) Flow cytometric analysis of natural killer (NK) cell subpopulations (CD56 bright /CD16 +/− and CD56 dim /CD16 + ) of patients’ peripheral blood mononuclear cells (PBMCs) and corresponding tumor tissues. PBMCs from healthy donors served as control. NK cell numbers were calculated and plotted as % of CD45 + lymphocytes ( n = 9 patients; n = 4 healthy controls). (D) Corresponding natural killer group 2D (NKG2D) expression levels of NK cell subpopulations plotted as mean fluorescent intensity (MFI) over background ( n = 9 patients). Statistical significance was assessed by one-way analysis of variance. ns, non-significant.

Techniques Used: Staining, Flow Cytometry, Expressing

Natural killer (NK) cell cytotoxicity and infiltration toward tumor spheroids is inhibited by shed NKG2DLs (sNKG2DLs) . (A) NK cells incubated with non-/sNKG2DL-depleted CAL27 supernatant (SN) or medium (kill CO) prior to co-incubation with FaDu, CAL27, or SiHa tumor spheroids (E:T ratio 5:1, 48 h). Tumor spheroid lysis was calculated by analysis of live (CFSE + /SytoxBlue − ) and dead (CFSE + /SytoxBlue + ) tumor cells. Bars correspond to mean ± SEM of eight tumor spheroids. (B) sNKG2DL levels of non-/depleted CAL27 SN analyzed by ELISA. Data are shown as mean ± SEM of duplicates. (C) NK cells treated with CAL27 SN, sMICA01, or untreated (kill CO) prior to coculturing with FaDu cells for 48 h. Representative phase-contrast pictures at 50× magnification are shown (size bar = 100 µm). Cryosections of spheroids were stained for NK cells (anti-CD45 antibody, red) and apoptosis (anti-active caspase-3 antibody, brown). Representative pictures are shown at 200× magnification (size bar = 200 µm). (D) Percentage of infiltrated NK cells in FaDu, CAL27, or SiHa tumor spheroids. Bars correspond to mean ± SEM of 10 vision fields. Statistical significance was assessed by one-way analysis of variance (A) and unpaired, two-tailed Student’s t -test (D) . ns, non-significant.
Figure Legend Snippet: Natural killer (NK) cell cytotoxicity and infiltration toward tumor spheroids is inhibited by shed NKG2DLs (sNKG2DLs) . (A) NK cells incubated with non-/sNKG2DL-depleted CAL27 supernatant (SN) or medium (kill CO) prior to co-incubation with FaDu, CAL27, or SiHa tumor spheroids (E:T ratio 5:1, 48 h). Tumor spheroid lysis was calculated by analysis of live (CFSE + /SytoxBlue − ) and dead (CFSE + /SytoxBlue + ) tumor cells. Bars correspond to mean ± SEM of eight tumor spheroids. (B) sNKG2DL levels of non-/depleted CAL27 SN analyzed by ELISA. Data are shown as mean ± SEM of duplicates. (C) NK cells treated with CAL27 SN, sMICA01, or untreated (kill CO) prior to coculturing with FaDu cells for 48 h. Representative phase-contrast pictures at 50× magnification are shown (size bar = 100 µm). Cryosections of spheroids were stained for NK cells (anti-CD45 antibody, red) and apoptosis (anti-active caspase-3 antibody, brown). Representative pictures are shown at 200× magnification (size bar = 200 µm). (D) Percentage of infiltrated NK cells in FaDu, CAL27, or SiHa tumor spheroids. Bars correspond to mean ± SEM of 10 vision fields. Statistical significance was assessed by one-way analysis of variance (A) and unpaired, two-tailed Student’s t -test (D) . ns, non-significant.

Techniques Used: Incubation, Lysis, Enzyme-linked Immunosorbent Assay, Staining, Two Tailed Test

5) Product Images from "Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction"

Article Title: Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction

Journal: Journal of Cancer

doi: 10.7150/jca.24415

Immunophenotypic characterization of human MSC and MSC-mediated increase in invasiveness of pancreatic cancer cells. A. FACS analysis of human MSC using anti-human CD34, CD45, CD73, and CD105. Mouse IgG as classmatch control for mouse monoclonal CD34, CD45, rabbit IgG for rabbit monoclonal CD73 and CD105. Indirect immunofluorescence using Alexa488-conjugated secondary antibodies. B and C. BxPC3 invasiveness was assessed under the co-culture condition with hMSCs by employing Boyden chamber method. Filter membrane set in the top chamber was sealed with Matrigel to close the micro pore. The mixed cells (BxPC3-GFP cells and hMSCs, ratio 1:1) were placed in the top chamber filled with serum free medium and serum was added or not added to the bottom well to be final concentrations 0.5% and 10%. After incubation for 12 hours, passed BxPC3-GFP cells through the filter membrane were observed by the fluorescence microscope (A) and their fluorescence intensities were measured and quantified (B). Data from A are means ± SD, *P
Figure Legend Snippet: Immunophenotypic characterization of human MSC and MSC-mediated increase in invasiveness of pancreatic cancer cells. A. FACS analysis of human MSC using anti-human CD34, CD45, CD73, and CD105. Mouse IgG as classmatch control for mouse monoclonal CD34, CD45, rabbit IgG for rabbit monoclonal CD73 and CD105. Indirect immunofluorescence using Alexa488-conjugated secondary antibodies. B and C. BxPC3 invasiveness was assessed under the co-culture condition with hMSCs by employing Boyden chamber method. Filter membrane set in the top chamber was sealed with Matrigel to close the micro pore. The mixed cells (BxPC3-GFP cells and hMSCs, ratio 1:1) were placed in the top chamber filled with serum free medium and serum was added or not added to the bottom well to be final concentrations 0.5% and 10%. After incubation for 12 hours, passed BxPC3-GFP cells through the filter membrane were observed by the fluorescence microscope (A) and their fluorescence intensities were measured and quantified (B). Data from A are means ± SD, *P

Techniques Used: FACS, Immunofluorescence, Co-Culture Assay, Incubation, Fluorescence, Microscopy

Immunohistological detection of MSCs in PDAC stroma. A. Antibodies for representative MSC markers, CD105, CD73, CD29 and αSMA were used to detect MSCs in PDAC stroma prepared from clinical specimens. Magnifications of the stained images were all x10. Left panels show hematoxylin and eosin (HE) staining (top x4; bottom x20). Circle shows a presence of fibroblast-like cells in the CD105 positive cells. B. Double immunofluorescence staining was performed in PDAC tissue sections for the different combinations as indicated in the images. MSC markers: CD105, CD73 and αSMA. Hematopoietic stem cell markers: CD34 and CD45.
Figure Legend Snippet: Immunohistological detection of MSCs in PDAC stroma. A. Antibodies for representative MSC markers, CD105, CD73, CD29 and αSMA were used to detect MSCs in PDAC stroma prepared from clinical specimens. Magnifications of the stained images were all x10. Left panels show hematoxylin and eosin (HE) staining (top x4; bottom x20). Circle shows a presence of fibroblast-like cells in the CD105 positive cells. B. Double immunofluorescence staining was performed in PDAC tissue sections for the different combinations as indicated in the images. MSC markers: CD105, CD73 and αSMA. Hematopoietic stem cell markers: CD34 and CD45.

Techniques Used: Staining, Double Immunofluorescence Staining

6) Product Images from "Augmenting Endogenous Levels of Retinal Annexin A1 Suppresses Uveitis in Mice"

Article Title: Augmenting Endogenous Levels of Retinal Annexin A1 Suppresses Uveitis in Mice

Journal: Translational Vision Science & Technology

doi: 10.1167/tvst.6.5.10

Local administration of hrAnx-A1 suppresses uveitis in mice. (A) Quantification by flow cytometry of neutrophils (CD45 + CD11b + Ly6G + ) infiltrating the eye at peak EIU disease (18 hours post induction) following administration of 2 μL PBS vehicle or hrAnxA1 (50 ng and 500 ng, respectively) by intravitreal injection. Data representative of two independent experiments, n = 5–7 mice ± SEM, **P
Figure Legend Snippet: Local administration of hrAnx-A1 suppresses uveitis in mice. (A) Quantification by flow cytometry of neutrophils (CD45 + CD11b + Ly6G + ) infiltrating the eye at peak EIU disease (18 hours post induction) following administration of 2 μL PBS vehicle or hrAnxA1 (50 ng and 500 ng, respectively) by intravitreal injection. Data representative of two independent experiments, n = 5–7 mice ± SEM, **P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Injection

Colocalization of AnxA1 with CD45 + leukocytes in human uveitis retinae and vitreous. AnxA1 and CD45 were stained by immunohistochemistry on retinal sections cut from eyes of uveitis patients. Scale bars: 50 μm.
Figure Legend Snippet: Colocalization of AnxA1 with CD45 + leukocytes in human uveitis retinae and vitreous. AnxA1 and CD45 were stained by immunohistochemistry on retinal sections cut from eyes of uveitis patients. Scale bars: 50 μm.

Techniques Used: Staining, Immunohistochemistry

7) Product Images from "The Role of Inflammation in β-cell Dedifferentiation"

Article Title: The Role of Inflammation in β-cell Dedifferentiation

Journal: Scientific Reports

doi: 10.1038/s41598-017-06731-w

Pancreatic islets of patients with type 2 diabetes contain more CD45 + immune cells than islets of non-diabetic subjects. ( a ) Representative islet histology of type 2 diabetic (T2D) and non-diabetic (ND) subjects. Peri-islet and intra-islet CD45 + immune cell count ( b and c , respectively) and distribution ( d and e , respectively) in human pancreatic tissue sections of T2D and ND control subjects. Islet area ( f ) and islet-area-corrected average CD45 + immune cell count ( g ) in T2D and ND control subjects. ( b – e ) n = 17, 16 for T2D and ND subjects, respectively, ( f , g ) n = 14, 10 for T2D and ND subjects. n = 30–35 islets/subject were counted. ***P
Figure Legend Snippet: Pancreatic islets of patients with type 2 diabetes contain more CD45 + immune cells than islets of non-diabetic subjects. ( a ) Representative islet histology of type 2 diabetic (T2D) and non-diabetic (ND) subjects. Peri-islet and intra-islet CD45 + immune cell count ( b and c , respectively) and distribution ( d and e , respectively) in human pancreatic tissue sections of T2D and ND control subjects. Islet area ( f ) and islet-area-corrected average CD45 + immune cell count ( g ) in T2D and ND control subjects. ( b – e ) n = 17, 16 for T2D and ND subjects, respectively, ( f , g ) n = 14, 10 for T2D and ND subjects. n = 30–35 islets/subject were counted. ***P

Techniques Used: Cell Counting

8) Product Images from "HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver"

Article Title: HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200317902

SDF-1 expression and engrafting cell accumulation within the liver. ( a – d ) NOD/SCID mice transplanted with CB CD34 + cells. ( a ) Hematoxylin and eosin staining 5–6 weeks after transplantation shows a bile duct (large arrow) adjacent to a large portal vein (pv) with cells surrounding the duct (dashed arrow) that are not observed in normal mouse liver. ( b ) Identification of SDF-1 in the bile ducts of NOD/SCID mouse liver. All epithelial cells in the bile ducts are strongly positive for SDF-1 (large arrow); scattered bile ductule cells (arrowheads) are also SDF-1–positive. ( c ) Human CD45 + hematopoietic cells (small arrows) are present in large numbers surrounding the bile ducts (large arrows) and accumulate to very high density between the ducts and the adjacent portal veins. ( d ) Human CD45 + cells are also observed as single cells or small clusters in random distribution in the hepatic sinusoids. Original magnification for a – d , ×400. ( e and f ) SDF-1 expression in normal adult human liver and in the liver of a patient with chronic hepatitis resulting from HCV infection was detected by immunohistochemistry. ( e ) Normal liver shows a single mature bile duct stained positive for SDF-1 (arrow) and absence of SDF-1 expression in endothelial cells lining a venous channel (arrowheads). ( f ) Liver from a patient with chronic liver disease from HCV infection shows extensive proliferation of bile ducts positive for SDF-1 expression (arrows) as well as expression of SDF-1 in bile ductule epithelium and/or canal of Hering or oval cells (arrowheads). Magnification for e and f , ×200.
Figure Legend Snippet: SDF-1 expression and engrafting cell accumulation within the liver. ( a – d ) NOD/SCID mice transplanted with CB CD34 + cells. ( a ) Hematoxylin and eosin staining 5–6 weeks after transplantation shows a bile duct (large arrow) adjacent to a large portal vein (pv) with cells surrounding the duct (dashed arrow) that are not observed in normal mouse liver. ( b ) Identification of SDF-1 in the bile ducts of NOD/SCID mouse liver. All epithelial cells in the bile ducts are strongly positive for SDF-1 (large arrow); scattered bile ductule cells (arrowheads) are also SDF-1–positive. ( c ) Human CD45 + hematopoietic cells (small arrows) are present in large numbers surrounding the bile ducts (large arrows) and accumulate to very high density between the ducts and the adjacent portal veins. ( d ) Human CD45 + cells are also observed as single cells or small clusters in random distribution in the hepatic sinusoids. Original magnification for a – d , ×400. ( e and f ) SDF-1 expression in normal adult human liver and in the liver of a patient with chronic hepatitis resulting from HCV infection was detected by immunohistochemistry. ( e ) Normal liver shows a single mature bile duct stained positive for SDF-1 (arrow) and absence of SDF-1 expression in endothelial cells lining a venous channel (arrowheads). ( f ) Liver from a patient with chronic liver disease from HCV infection shows extensive proliferation of bile ducts positive for SDF-1 expression (arrows) as well as expression of SDF-1 in bile ductule epithelium and/or canal of Hering or oval cells (arrowheads). Magnification for e and f , ×200.

Techniques Used: Expressing, Mouse Assay, Staining, Transplantation Assay, Infection, Immunohistochemistry

9) Product Images from "Atrial inflammation in different atrial fibrillation subtypes and its relation with clinical risk factors"

Article Title: Atrial inflammation in different atrial fibrillation subtypes and its relation with clinical risk factors

Journal: Clinical Research in Cardiology

doi: 10.1007/s00392-020-01619-8

The atrial inflammatory cells infiltrate in diabetic versus non-diabetic AF patients. The number of CD45+ ( a ) and CD3+ ( b ) cells in the myocardium and adipose tissue in paroxysmal (PAR) and long-standing persistent/permanent (LS-PE/PER) AF patients with ( n = 8) or without DM ( n = 42). Data are presented as box plot with median and min–max percentiles (whiskers). Bars represent mean ± SD
Figure Legend Snippet: The atrial inflammatory cells infiltrate in diabetic versus non-diabetic AF patients. The number of CD45+ ( a ) and CD3+ ( b ) cells in the myocardium and adipose tissue in paroxysmal (PAR) and long-standing persistent/permanent (LS-PE/PER) AF patients with ( n = 8) or without DM ( n = 42). Data are presented as box plot with median and min–max percentiles (whiskers). Bars represent mean ± SD

Techniques Used:

The atrial inflammatory cell infiltrate in AF patients of different age. The number of CD45+ a cells in the myocardium and adipose tissue in paroxysmal (PAR) and long-standing persistent/permanent (LS-PE/PER) AF patients aged below 55 years old (
Figure Legend Snippet: The atrial inflammatory cell infiltrate in AF patients of different age. The number of CD45+ a cells in the myocardium and adipose tissue in paroxysmal (PAR) and long-standing persistent/permanent (LS-PE/PER) AF patients aged below 55 years old (

Techniques Used:

The atrial inflammatory cell infiltrate in male versus female AF patients. The number of CD45+ a cells in the myocardium and adipose tissue of male ( n = 36) and female ( n = 14) patients with paroxysmal (PAR) and long-standing persistent/permanent (LS-PE/PER) AF. The number of CD3+ b cells in the myocardium and adipose tissue of male (M) and female (F) patients with AF. Data are presented as box plot with median and min–max percentiles (whiskers). Bars represent mean ± SD
Figure Legend Snippet: The atrial inflammatory cell infiltrate in male versus female AF patients. The number of CD45+ a cells in the myocardium and adipose tissue of male ( n = 36) and female ( n = 14) patients with paroxysmal (PAR) and long-standing persistent/permanent (LS-PE/PER) AF. The number of CD3+ b cells in the myocardium and adipose tissue of male (M) and female (F) patients with AF. Data are presented as box plot with median and min–max percentiles (whiskers). Bars represent mean ± SD

Techniques Used:

Inflammatory cells in the atria of patients with AF. An example of CD45+ ( a ) and CD3+ ( b ) cells (black arrows) in the atria of patients with AF. M myocardium, A adipose tissue, scale bar 50 µm. The number of CD45+ and CD3+ cells/mm 2 in the myocardium (Myo) and adipose tissue (Adi) in the atria of control patients without AF (Con), patients with paroxysmal (PAR), long-standing persistent/permanent (LS-PE/PER) AF. ( c ) Hematoxylin–eosin stained cross section of the left atrial wall of an AF patient showing the spatial distribution of adipose tissue (A) and myocardium (M). Scar bar 2 mm. Data are presented as box plot with median and min–max percentiles (whiskers). Bars represent mean ± SD. ΔΔΔ means compared with myocardium of control group; ### means compared with adipose tissue of the control group. *** p
Figure Legend Snippet: Inflammatory cells in the atria of patients with AF. An example of CD45+ ( a ) and CD3+ ( b ) cells (black arrows) in the atria of patients with AF. M myocardium, A adipose tissue, scale bar 50 µm. The number of CD45+ and CD3+ cells/mm 2 in the myocardium (Myo) and adipose tissue (Adi) in the atria of control patients without AF (Con), patients with paroxysmal (PAR), long-standing persistent/permanent (LS-PE/PER) AF. ( c ) Hematoxylin–eosin stained cross section of the left atrial wall of an AF patient showing the spatial distribution of adipose tissue (A) and myocardium (M). Scar bar 2 mm. Data are presented as box plot with median and min–max percentiles (whiskers). Bars represent mean ± SD. ΔΔΔ means compared with myocardium of control group; ### means compared with adipose tissue of the control group. *** p

Techniques Used: Staining

10) Product Images from "Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice"

Article Title: Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004291

Pre-depletion of pDC reduces HIV-R3A induced death of human leukocytes. ( A ) CD4 T cell (CD3 + CD8 − ) counts in peripheral blood (PBL) and spleens (SP) of mock or HIV-R3A infected mice. ( B ) CD8 T cell (CD3 + CD4 − CD8 + ) counts in PBL and SP. ( C ) Human CD45 + leukocyte counts in PBL and SP. ( D ) Representative histograms show percentages of dead CD4 T cells, CD8 T cells and huCD45 + cells in spleens of mice infected with mock or R3A at 8 dpi. ( E ) Summarized data of D. mock, n = 6; isotype+R3A, n = 9; 15B+R3A, n = 12. Bars in all graphs indicate median value. * and ** indicate p
Figure Legend Snippet: Pre-depletion of pDC reduces HIV-R3A induced death of human leukocytes. ( A ) CD4 T cell (CD3 + CD8 − ) counts in peripheral blood (PBL) and spleens (SP) of mock or HIV-R3A infected mice. ( B ) CD8 T cell (CD3 + CD4 − CD8 + ) counts in PBL and SP. ( C ) Human CD45 + leukocyte counts in PBL and SP. ( D ) Representative histograms show percentages of dead CD4 T cells, CD8 T cells and huCD45 + cells in spleens of mice infected with mock or R3A at 8 dpi. ( E ) Summarized data of D. mock, n = 6; isotype+R3A, n = 9; 15B+R3A, n = 12. Bars in all graphs indicate median value. * and ** indicate p

Techniques Used: Infection, Mouse Assay

Depletion of pDC during chronic infection reduces HIV-1 induced immunopathogenesis. Humanized mice were infected HIV-JRCSF and were treated weekly with 15B or control at 11 weeks post-infection and terminated at 21 weeks post-infection (mock, n = 6; JR-CSF+control, n = 9; JR-CSF+15B, n = 9). ( A–C ) Summarized data show relative numbers in PBL and SP of ( A ) CD4 T cells (CD3 + CD8 − ), ( B ) CD8 T cells (CD3 + CD4 − CD8 + ), and ( C ) human CD45 + leukocytes. ( D ) Immunohistochemistry staining for human CD45 + cells in spleens of mock or HIV-1 infected mice. ( E ) FACS plots of cell death in human CD4 + T cells, CD8 + T cells and huCD45 + leukocytes from spleens. ( F ) Summary data show percentages of cell death in CD4 + T cells, CD8 + T cells and huCD45 + leukocytes. Bars in dot graphs indicate median value. * and ** indicate p
Figure Legend Snippet: Depletion of pDC during chronic infection reduces HIV-1 induced immunopathogenesis. Humanized mice were infected HIV-JRCSF and were treated weekly with 15B or control at 11 weeks post-infection and terminated at 21 weeks post-infection (mock, n = 6; JR-CSF+control, n = 9; JR-CSF+15B, n = 9). ( A–C ) Summarized data show relative numbers in PBL and SP of ( A ) CD4 T cells (CD3 + CD8 − ), ( B ) CD8 T cells (CD3 + CD4 − CD8 + ), and ( C ) human CD45 + leukocytes. ( D ) Immunohistochemistry staining for human CD45 + cells in spleens of mock or HIV-1 infected mice. ( E ) FACS plots of cell death in human CD4 + T cells, CD8 + T cells and huCD45 + leukocytes from spleens. ( F ) Summary data show percentages of cell death in CD4 + T cells, CD8 + T cells and huCD45 + leukocytes. Bars in dot graphs indicate median value. * and ** indicate p

Techniques Used: Infection, Mouse Assay, Immunohistochemistry, Staining, FACS

Pre-infection depletion of pDC abolishes IFN-I induction during acute HIV-1 infection in humanized mice. ( A ) Summarized data of pDC percentages in total human leukocytes (CD45 + ) from humanized mice are shown mice. Mice were treated with either 15B or isotype control (iso) antibody. After pDC depletion, mice were infected with HIV-R3A and terminated on 8 days post infection (dpi) for analysis. Mock infected mice, n = 6; isotype+R3A infected mice, n = 9; 15B+R3A infected mice, n = 12. ( B ) Plasma levels of IFN-α2 from mock, HIV-1 infected and 15B or isotype mAb treated mice were quantified by Luminex assays. Mock, n = 3; isotype+R3A, n = 5; 15B+R3A, n = 5. ( C ) The mRNA expression of major type I IFN genes in purified human cells (CD45 + ) from mouse spleens was measured by real-time PCR. ( D ) ISGs (Mx1 and TRIM22) expression in purified human cells (CD45 + ) from mouse spleens was measured by real-time PCR. Mock, n = 3; isotype+R3A, n = 5; 15B+R3A, n = 5. Mice were analyzed at 8 days post infection. Error bars in graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p
Figure Legend Snippet: Pre-infection depletion of pDC abolishes IFN-I induction during acute HIV-1 infection in humanized mice. ( A ) Summarized data of pDC percentages in total human leukocytes (CD45 + ) from humanized mice are shown mice. Mice were treated with either 15B or isotype control (iso) antibody. After pDC depletion, mice were infected with HIV-R3A and terminated on 8 days post infection (dpi) for analysis. Mock infected mice, n = 6; isotype+R3A infected mice, n = 9; 15B+R3A infected mice, n = 12. ( B ) Plasma levels of IFN-α2 from mock, HIV-1 infected and 15B or isotype mAb treated mice were quantified by Luminex assays. Mock, n = 3; isotype+R3A, n = 5; 15B+R3A, n = 5. ( C ) The mRNA expression of major type I IFN genes in purified human cells (CD45 + ) from mouse spleens was measured by real-time PCR. ( D ) ISGs (Mx1 and TRIM22) expression in purified human cells (CD45 + ) from mouse spleens was measured by real-time PCR. Mock, n = 3; isotype+R3A, n = 5; 15B+R3A, n = 5. Mice were analyzed at 8 days post infection. Error bars in graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p

Techniques Used: Infection, Mouse Assay, Luminex, Expressing, Purification, Real-time Polymerase Chain Reaction

Depletion of pDC during chronic HIV-1 infection increases HIV-1 replication associated with reduced IFN-I expression. Humanized mice were infected HIV-JRCSF and were treated weekly with 15B or control at 11 weeks post-infection and terminated at 21 weeks post-infection (mock, n = 6; JR-CSF+control, n = 9; JR-CSF+15B, n = 9). ( A ) Plasma HIV-1 RNA levels (genome copy#×log10/ml) at each time point were analyzed by real-time PCR. ( B ) Representative FACS histograms and summarized data ( C ) show percentages of HIV p24-positive CD4 T cells (CD3 + CD8 − ) in the spleen. ( D ) The production of IFNα2 in the plasma, from mock infected (n = 4), JR-CSF+PBS (n = 5) and JR-CSF+15B (n = 5) at either 11 wpi (pre-) or 21 wpi (post-), was measured by Luminex. ( E ) The mRNA levels of IFN-I genes or ISGs Mx1 and TRIM22 ( F ) in purified human CD45 + cells from spleens (n = 5). All bars in dot graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p
Figure Legend Snippet: Depletion of pDC during chronic HIV-1 infection increases HIV-1 replication associated with reduced IFN-I expression. Humanized mice were infected HIV-JRCSF and were treated weekly with 15B or control at 11 weeks post-infection and terminated at 21 weeks post-infection (mock, n = 6; JR-CSF+control, n = 9; JR-CSF+15B, n = 9). ( A ) Plasma HIV-1 RNA levels (genome copy#×log10/ml) at each time point were analyzed by real-time PCR. ( B ) Representative FACS histograms and summarized data ( C ) show percentages of HIV p24-positive CD4 T cells (CD3 + CD8 − ) in the spleen. ( D ) The production of IFNα2 in the plasma, from mock infected (n = 4), JR-CSF+PBS (n = 5) and JR-CSF+15B (n = 5) at either 11 wpi (pre-) or 21 wpi (post-), was measured by Luminex. ( E ) The mRNA levels of IFN-I genes or ISGs Mx1 and TRIM22 ( F ) in purified human CD45 + cells from spleens (n = 5). All bars in dot graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p

Techniques Used: Infection, Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, FACS, Luminex, Purification

Specific depletion of human pDC in lymphoid organs in vivo with a human pDC-reactive monoclonal antibody. Humanized Mice were treated with either 15B or isotype control (iso) antibody for 3 times on days -5, -3, -1 prior to termination. Percentages of pDC (Lin − CD4 + CD123 + ) in total human leukocytes (CD45 + ) are analyzed. ( A ) Representative FACS plots and summarized data show relative pDC frequencies before and after antibody treatment in the peripheral blood (n = 7). ( B ) Representative FACS plots and summarized data show pDC depletion by 15B in mesenteric lymph nodes (mLN) and spleen (SP, isotype n = 4; 15B n = 5). All bars in dot graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p
Figure Legend Snippet: Specific depletion of human pDC in lymphoid organs in vivo with a human pDC-reactive monoclonal antibody. Humanized Mice were treated with either 15B or isotype control (iso) antibody for 3 times on days -5, -3, -1 prior to termination. Percentages of pDC (Lin − CD4 + CD123 + ) in total human leukocytes (CD45 + ) are analyzed. ( A ) Representative FACS plots and summarized data show relative pDC frequencies before and after antibody treatment in the peripheral blood (n = 7). ( B ) Representative FACS plots and summarized data show pDC depletion by 15B in mesenteric lymph nodes (mLN) and spleen (SP, isotype n = 4; 15B n = 5). All bars in dot graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p

Techniques Used: In Vivo, Mouse Assay, FACS

11) Product Images from "Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction"

Article Title: Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction

Journal: Journal of Cancer

doi: 10.7150/jca.24415

Immunophenotypic characterization of human MSC and MSC-mediated increase in invasiveness of pancreatic cancer cells. A. FACS analysis of human MSC using anti-human CD34, CD45, CD73, and CD105. Mouse IgG as classmatch control for mouse monoclonal CD34, CD45, rabbit IgG for rabbit monoclonal CD73 and CD105. Indirect immunofluorescence using Alexa488-conjugated secondary antibodies. B and C. BxPC3 invasiveness was assessed under the co-culture condition with hMSCs by employing Boyden chamber method. Filter membrane set in the top chamber was sealed with Matrigel to close the micro pore. The mixed cells (BxPC3-GFP cells and hMSCs, ratio 1:1) were placed in the top chamber filled with serum free medium and serum was added or not added to the bottom well to be final concentrations 0.5% and 10%. After incubation for 12 hours, passed BxPC3-GFP cells through the filter membrane were observed by the fluorescence microscope (A) and their fluorescence intensities were measured and quantified (B). Data from A are means ± SD, *P
Figure Legend Snippet: Immunophenotypic characterization of human MSC and MSC-mediated increase in invasiveness of pancreatic cancer cells. A. FACS analysis of human MSC using anti-human CD34, CD45, CD73, and CD105. Mouse IgG as classmatch control for mouse monoclonal CD34, CD45, rabbit IgG for rabbit monoclonal CD73 and CD105. Indirect immunofluorescence using Alexa488-conjugated secondary antibodies. B and C. BxPC3 invasiveness was assessed under the co-culture condition with hMSCs by employing Boyden chamber method. Filter membrane set in the top chamber was sealed with Matrigel to close the micro pore. The mixed cells (BxPC3-GFP cells and hMSCs, ratio 1:1) were placed in the top chamber filled with serum free medium and serum was added or not added to the bottom well to be final concentrations 0.5% and 10%. After incubation for 12 hours, passed BxPC3-GFP cells through the filter membrane were observed by the fluorescence microscope (A) and their fluorescence intensities were measured and quantified (B). Data from A are means ± SD, *P

Techniques Used: FACS, Immunofluorescence, Co-Culture Assay, Incubation, Fluorescence, Microscopy

Immunohistological detection of MSCs in PDAC stroma. A. Antibodies for representative MSC markers, CD105, CD73, CD29 and αSMA were used to detect MSCs in PDAC stroma prepared from clinical specimens. Magnifications of the stained images were all x10. Left panels show hematoxylin and eosin (HE) staining (top x4; bottom x20). Circle shows a presence of fibroblast-like cells in the CD105 positive cells. B. Double immunofluorescence staining was performed in PDAC tissue sections for the different combinations as indicated in the images. MSC markers: CD105, CD73 and αSMA. Hematopoietic stem cell markers: CD34 and CD45.
Figure Legend Snippet: Immunohistological detection of MSCs in PDAC stroma. A. Antibodies for representative MSC markers, CD105, CD73, CD29 and αSMA were used to detect MSCs in PDAC stroma prepared from clinical specimens. Magnifications of the stained images were all x10. Left panels show hematoxylin and eosin (HE) staining (top x4; bottom x20). Circle shows a presence of fibroblast-like cells in the CD105 positive cells. B. Double immunofluorescence staining was performed in PDAC tissue sections for the different combinations as indicated in the images. MSC markers: CD105, CD73 and αSMA. Hematopoietic stem cell markers: CD34 and CD45.

Techniques Used: Staining, Double Immunofluorescence Staining

Related Articles

Activation Assay:

Article Title: Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction
Article Snippet: Human PDAC lines, AsPC1, MiaPaCa-2, and Panc-1, all derived from the patients with pancreatic invasive ductal adenocarcinoma were obtained from ATCC (Manassas, VA) and maintained with RPMI medium (Wako Laboratory Chemicals, Japan) supplemented with 10% FBS for the analysis. .. Antibodies used in the present study were as follows: mouse anti-human CD105 antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-human CD73 antibody (NT5E/CD73 (D7F9A); Cell Signaling Technology Japan; cross-reactive to mouse, rat), rabbit anti-human CD29 antibody (Cell Signaling Technology Japan), mouse anti-human αSMA antibody (Dako, Agilent Technologies, Japan), mouse anti-human CD34 antibody (Dako, Agilent Technologies, Japan), mouse anti-human CD45, LCA antibody (Dako, Agilent Technologies, Japan), rabbit anti-human MMP-3 antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-human MMP-9 antibody (Cell Signaling Technology Japan), mouse anti-human AREG antibody (R & D Systems, Minneapolis, MN), mouse anti-human EGFR antibody (NICHIREI BIOSCIENCES INC, Japan), sheep anti-human Fibroblast activation protein alpha (FAPa) antibody (R & D systems, Minneapolis, MN). .. Flow Cytometric Analysis.

Staining:

Article Title: Toll-like receptor 2/4 inhibitors can reduce preterm birth in mice
Article Snippet: Sections were stained using anti-CD25 (1:100, Abcam, ab128955, Cambridge, MA, USA) and anti-FOXP3 (1:1000, Santa Cruz Biotechnology, 2A11G9, Dallas, TX, USA) primary antibodies overnight at 4°C. .. Mouse anti-human CD45 (1: 100, Dako, 00087397, Santa Clara, CA, USA) antibody was used to stain lymphocytes. .. For FOXP3/CD25, horseradish peroxidase-conjugated anti-mouse and alkaline phosphatase-conjugated anti-rabbit (Biocare Medical, MRCT525H, Pacheco, CA, USA) secondary antibodies were used.

Article Title: In vitro and in vivo efficacy of an anti-CD203c conjugated antibody (AGS-16C3F) in mouse models of advanced systemic mastocytosis
Article Snippet: .. Fixed tissues were stained with a mouse anti-human CD45 mAb (Dako, Trappes, France), as previously described. .. Staining was visualized by Histomouse Kit (Thermo Fisher).

Article Title: Leukemia regression by vascular disruption and antiangiogenic therapy
Article Snippet: When dual staining with MECA and HIF 1α, it was possible to apply the 2 antibodies simultaneously and incubate as a cocktail. .. Staining with mouse anti–human CD45 (1:50; DakoCytomation) required the use of a mouse on mouse (M.O.M.) .. Standard Kit (Vector Laboratories).

Article Title: Atrial inflammation in different atrial fibrillation subtypes and its relation with clinical risk factors
Article Snippet: .. To block endogenous peroxidase activity, the sections were incubated in 0.3% H2 O2 in methanol for 30 min for staining with rabbit–anti-human CD3 (T lymphocytes; Dako Agilent, Amstelveen, The Netherlands), antigen retrieval was performed by boiling the sections in Tris–EDTA buffer (10 mM, pH 9.0) for 10 min. No antigen retrieval was used for staining with mouse anti-human CD45 (lymphocytes; Dako Agilent). .. The sections were washed with phosphate-buffered saline (PBS) and then, incubated with the primary antibodies against CD45 or CD3, both diluted 1:50 in normal antibody diluent (Dako Agilent) for 1 h at room temperature.

Incubation:

Article Title: High CTLA-4 expression correlates with poor prognosis in thymoma patients
Article Snippet: After 2 washes in PBS sections were treated with 3% H2 O2 for 20 min, washed, treated for 6 min with potassium permanganate solution (KMnO4 0.06%) and incubated for 1 h at room temperature with 3% bovine serum albumin and 0.3% Triton X-100 in PBS. .. After that sections were incubated overnight at 4°C with mouse anti-CTLA-4 mAb (Novus Biologicals, Littleton, USA, clone BNI3, isotype IgG2a, 1:25) followed by anti-mouse IgG AlexaFluor 488 secondary Ab (Thermo Fisher Scientific corporation, Waltham, USA, 1:100) for 60 min at 37°C, then overnight at 4°C with mouse anti-human CD45 mAb (Dako, Glostrup, Denmark, clone 2B11+PD7/26, isotype IgG1, 1:50) followed by anti-mouse IgG1 AlexaFluor 594 secondary Ab (Thermo Fisher Scientific corporation, 1:100) for 60 min at 37°C. .. In some experiments sections were incubated overnight at 4°C with mouse anti-CTLA-4 mAb (1:25) followed by anti-mouse IgG2a AlexaFluor 594 secondary Ab (Thermo Fisher Scientific corporation, 1:100) for 60 min at 37°C, then overnight at 4°C with mouse anti-human Vimentin mAb (Sigma, Saint Louis, USA, clone V9, isotype IgG1, 1:50) followed by anti-mouse IgG AlexaFluor 488 secondary Ab (Thermo Fisher Scientific corporation, 1:100) for 60 min at 37°C.

Article Title: Atrial inflammation in different atrial fibrillation subtypes and its relation with clinical risk factors
Article Snippet: .. To block endogenous peroxidase activity, the sections were incubated in 0.3% H2 O2 in methanol for 30 min for staining with rabbit–anti-human CD3 (T lymphocytes; Dako Agilent, Amstelveen, The Netherlands), antigen retrieval was performed by boiling the sections in Tris–EDTA buffer (10 mM, pH 9.0) for 10 min. No antigen retrieval was used for staining with mouse anti-human CD45 (lymphocytes; Dako Agilent). .. The sections were washed with phosphate-buffered saline (PBS) and then, incubated with the primary antibodies against CD45 or CD3, both diluted 1:50 in normal antibody diluent (Dako Agilent) for 1 h at room temperature.

Blocking Assay:

Article Title: Atrial inflammation in different atrial fibrillation subtypes and its relation with clinical risk factors
Article Snippet: .. To block endogenous peroxidase activity, the sections were incubated in 0.3% H2 O2 in methanol for 30 min for staining with rabbit–anti-human CD3 (T lymphocytes; Dako Agilent, Amstelveen, The Netherlands), antigen retrieval was performed by boiling the sections in Tris–EDTA buffer (10 mM, pH 9.0) for 10 min. No antigen retrieval was used for staining with mouse anti-human CD45 (lymphocytes; Dako Agilent). .. The sections were washed with phosphate-buffered saline (PBS) and then, incubated with the primary antibodies against CD45 or CD3, both diluted 1:50 in normal antibody diluent (Dako Agilent) for 1 h at room temperature.

Activity Assay:

Article Title: Atrial inflammation in different atrial fibrillation subtypes and its relation with clinical risk factors
Article Snippet: .. To block endogenous peroxidase activity, the sections were incubated in 0.3% H2 O2 in methanol for 30 min for staining with rabbit–anti-human CD3 (T lymphocytes; Dako Agilent, Amstelveen, The Netherlands), antigen retrieval was performed by boiling the sections in Tris–EDTA buffer (10 mM, pH 9.0) for 10 min. No antigen retrieval was used for staining with mouse anti-human CD45 (lymphocytes; Dako Agilent). .. The sections were washed with phosphate-buffered saline (PBS) and then, incubated with the primary antibodies against CD45 or CD3, both diluted 1:50 in normal antibody diluent (Dako Agilent) for 1 h at room temperature.

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    Agilent technologies anti human cd45 antibody
    Efficacy of Dynole 34-2 against LSCs from primary Lmo2 -transgenic T-ALL. a Flow sorting and schematic representation of the transplantation strategy of purified leukemic populations from three different primary Lmo2 Tg thymomas. Indicated leukemic populations were purified and 5 × 10 4 cells were transplanted into sublethally irradiated <t>Cd45.1</t> + recipients. b Kaplan–Meier curves of mice injected with the purified populations (DN1, n = 5; DN3a, n = 17; DN4, n = 15; ISP8, n = 10; DP, n = 12) from three different Lmo2 Tg primary leukemias. c Relative levels of activated Stat5 (pStat5) and Hes1 in leukemic DN3a cells from primary Lmo2 Tg T-ALL treated with Dynole 34-2, after in vitro stimulation of either the IL-7 (left) or Notch1 (right) signalling pathway. Primary leukemias are indicated on the right with T-ALL 249 in blue, T-ALL 582 in green, T-ALL 573 in orange, and other leukemias are in black; unstimulated leukemic DN3a cells treated with vehicle were used as control, and reported as 1 (dashed line). Mean ± SEM ( N = 6 primary leukemias, performed in duplicate), two-way ANOVA with Bonferroni correction test; ** P
    Anti Human Cd45 Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunohistochemistry in patient 2. S100 immunopositive nerve fibres are present in the media ( A ) and <t>CD45</t> immunopositive leukocytes are few and scattered in the vein wall ( B ). Scale bar = 30 μm in A and B
    Cd45 Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies mouse anti human cd45 antibody
    In situ CXCL10 expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Combined insulin, glucagon, <t>CD45,</t> and CXCL10 stains were performed as detailed in RESEARCH DESIGN AND METHODS . Note the absence of CXCL10 staining in the healthy control subjects (case identification no. 6112) but ready presence in type 1 diabetic samples (identification nos. 6087 and 6052), sometimes in close association with infiltrating leukocytes (CD45 + ). To confirm the pattern of exocrine CXCL10 staining, we included a sample from an additional diabetic donor with clinically confirmed pancreatitis (identification no. 6036). (A high-quality digital representation of this figure is available in the online issue.)
    Mouse Anti Human Cd45 Antibody, supplied by Agilent technologies, 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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    Host-derived IL33 activates pancreatic T cell immunity. ( a ) Gene set enrichment analysis of bulk RNA-seq from purified <t>CD45+</t> immune cells from Il33 +/+ and Il33 −/− PDAC mice. Enrichment plots and enrichment scores are shown for three gene sets comparing expression in Il33 −/− to Il33 +/+ (n=3 mice/group). FDR, false discovery rate. ( b ) Gating of CD8 + T cells and ( c ) frequencies of various immune cell types ( left ) and CD4 + T cell lineages ( right ) in Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( d ) Frequency of T central memory (T cm ) cells (CD45 + CD3 + CD8 + CD44 + CD62L + ) in tumor draining lymph nodes and non-tumor draining distant lymphoid organs (inguinal lymph node and spleen) in Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( e ) Frequency of CD8 + T cells in subcutaneous PDAC tumors. DC, dendritic cells; MDSC, myeloid-derived suppressor cells; NK, natural killer cells; NKT, natural killer T cells; T reg , regulatory T cells. Data were analyzed 14 days post tumor implantation or at the time points indicated. Horizontal bars mark medians, error bars mark s.e.m. n indicates individual mice analyzed separately in at least two independent experiments with n≥2/group. P values determined by one-way ANOVA ( d ).
    Anti Cd45 Antibodies, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Efficacy of Dynole 34-2 against LSCs from primary Lmo2 -transgenic T-ALL. a Flow sorting and schematic representation of the transplantation strategy of purified leukemic populations from three different primary Lmo2 Tg thymomas. Indicated leukemic populations were purified and 5 × 10 4 cells were transplanted into sublethally irradiated Cd45.1 + recipients. b Kaplan–Meier curves of mice injected with the purified populations (DN1, n = 5; DN3a, n = 17; DN4, n = 15; ISP8, n = 10; DP, n = 12) from three different Lmo2 Tg primary leukemias. c Relative levels of activated Stat5 (pStat5) and Hes1 in leukemic DN3a cells from primary Lmo2 Tg T-ALL treated with Dynole 34-2, after in vitro stimulation of either the IL-7 (left) or Notch1 (right) signalling pathway. Primary leukemias are indicated on the right with T-ALL 249 in blue, T-ALL 582 in green, T-ALL 573 in orange, and other leukemias are in black; unstimulated leukemic DN3a cells treated with vehicle were used as control, and reported as 1 (dashed line). Mean ± SEM ( N = 6 primary leukemias, performed in duplicate), two-way ANOVA with Bonferroni correction test; ** P

    Journal: Nature Communications

    Article Title: Small molecule inhibition of Dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells

    doi: 10.1038/s41467-020-20091-6

    Figure Lengend Snippet: Efficacy of Dynole 34-2 against LSCs from primary Lmo2 -transgenic T-ALL. a Flow sorting and schematic representation of the transplantation strategy of purified leukemic populations from three different primary Lmo2 Tg thymomas. Indicated leukemic populations were purified and 5 × 10 4 cells were transplanted into sublethally irradiated Cd45.1 + recipients. b Kaplan–Meier curves of mice injected with the purified populations (DN1, n = 5; DN3a, n = 17; DN4, n = 15; ISP8, n = 10; DP, n = 12) from three different Lmo2 Tg primary leukemias. c Relative levels of activated Stat5 (pStat5) and Hes1 in leukemic DN3a cells from primary Lmo2 Tg T-ALL treated with Dynole 34-2, after in vitro stimulation of either the IL-7 (left) or Notch1 (right) signalling pathway. Primary leukemias are indicated on the right with T-ALL 249 in blue, T-ALL 582 in green, T-ALL 573 in orange, and other leukemias are in black; unstimulated leukemic DN3a cells treated with vehicle were used as control, and reported as 1 (dashed line). Mean ± SEM ( N = 6 primary leukemias, performed in duplicate), two-way ANOVA with Bonferroni correction test; ** P

    Article Snippet: Samples were incubated with an anti-human CD45 antibody (Dako, #M0701) in Dako Autostainer Plus solution, then incubated with a sheep anti-mouse IgG conjugated to HRP and processed as per standard protocols using 3,3’ Diaminobenzidine staining (#ab64238, Abcam) for all immunoblots, as previously described .

    Techniques: Transgenic Assay, Transplantation Assay, Purification, Irradiation, Mouse Assay, Injection, In Vitro

    Immunohistochemistry in patient 2. S100 immunopositive nerve fibres are present in the media ( A ) and CD45 immunopositive leukocytes are few and scattered in the vein wall ( B ). Scale bar = 30 μm in A and B

    Journal: The International Journal of Angiology : Official Publication of the International College of Angiology, Inc

    Article Title: Evidence for unmyelinated C fibres and inflammatory cells in human varicose saphenous vein

    doi:

    Figure Lengend Snippet: Immunohistochemistry in patient 2. S100 immunopositive nerve fibres are present in the media ( A ) and CD45 immunopositive leukocytes are few and scattered in the vein wall ( B ). Scale bar = 30 μm in A and B

    Article Snippet: On deparaffinized sections using the standard peroxidase-antiperoxidase technique, immunostaining was performed with protein S100 antibody (polyclonal rabbit anti-S100; DakoCytomation, Denmark A/S) for the labelling of Schwann cells (ie, nerve fibres) and CD45 antibody (monoclonal mouse antihuman CD45, clones 2B11 + PD7/26, DakoCytomation) for the labelling of panleukocyte cells.

    Techniques: Immunohistochemistry

    In situ CXCL10 expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Combined insulin, glucagon, CD45, and CXCL10 stains were performed as detailed in RESEARCH DESIGN AND METHODS . Note the absence of CXCL10 staining in the healthy control subjects (case identification no. 6112) but ready presence in type 1 diabetic samples (identification nos. 6087 and 6052), sometimes in close association with infiltrating leukocytes (CD45 + ). To confirm the pattern of exocrine CXCL10 staining, we included a sample from an additional diabetic donor with clinically confirmed pancreatitis (identification no. 6036). (A high-quality digital representation of this figure is available in the online issue.)

    Journal: Diabetes

    Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

    doi: 10.2337/db11-0853

    Figure Lengend Snippet: In situ CXCL10 expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Combined insulin, glucagon, CD45, and CXCL10 stains were performed as detailed in RESEARCH DESIGN AND METHODS . Note the absence of CXCL10 staining in the healthy control subjects (case identification no. 6112) but ready presence in type 1 diabetic samples (identification nos. 6087 and 6052), sometimes in close association with infiltrating leukocytes (CD45 + ). To confirm the pattern of exocrine CXCL10 staining, we included a sample from an additional diabetic donor with clinically confirmed pancreatitis (identification no. 6036). (A high-quality digital representation of this figure is available in the online issue.)

    Article Snippet: Endogenous peroxidase was quenched with 3% hydrogen peroxide for 30 min, slides were blocked for 1 h in Tris-NaCl Buffer (TNB) buffer (Zymed) and incubated with mouse anti-human CD45 antibody (Richard Allen Scientific) for 2 h, and CD45-labeled cells were visualized by immunoperoxidase reaction with diaminobezidine (Dako).

    Techniques: In Situ, Expressing, Staining

    Host-derived IL33 activates pancreatic T cell immunity. ( a ) Gene set enrichment analysis of bulk RNA-seq from purified CD45+ immune cells from Il33 +/+ and Il33 −/− PDAC mice. Enrichment plots and enrichment scores are shown for three gene sets comparing expression in Il33 −/− to Il33 +/+ (n=3 mice/group). FDR, false discovery rate. ( b ) Gating of CD8 + T cells and ( c ) frequencies of various immune cell types ( left ) and CD4 + T cell lineages ( right ) in Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( d ) Frequency of T central memory (T cm ) cells (CD45 + CD3 + CD8 + CD44 + CD62L + ) in tumor draining lymph nodes and non-tumor draining distant lymphoid organs (inguinal lymph node and spleen) in Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( e ) Frequency of CD8 + T cells in subcutaneous PDAC tumors. DC, dendritic cells; MDSC, myeloid-derived suppressor cells; NK, natural killer cells; NKT, natural killer T cells; T reg , regulatory T cells. Data were analyzed 14 days post tumor implantation or at the time points indicated. Horizontal bars mark medians, error bars mark s.e.m. n indicates individual mice analyzed separately in at least two independent experiments with n≥2/group. P values determined by one-way ANOVA ( d ).

    Journal: Nature

    Article Title: ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity

    doi: 10.1038/s41586-020-2015-4

    Figure Lengend Snippet: Host-derived IL33 activates pancreatic T cell immunity. ( a ) Gene set enrichment analysis of bulk RNA-seq from purified CD45+ immune cells from Il33 +/+ and Il33 −/− PDAC mice. Enrichment plots and enrichment scores are shown for three gene sets comparing expression in Il33 −/− to Il33 +/+ (n=3 mice/group). FDR, false discovery rate. ( b ) Gating of CD8 + T cells and ( c ) frequencies of various immune cell types ( left ) and CD4 + T cell lineages ( right ) in Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( d ) Frequency of T central memory (T cm ) cells (CD45 + CD3 + CD8 + CD44 + CD62L + ) in tumor draining lymph nodes and non-tumor draining distant lymphoid organs (inguinal lymph node and spleen) in Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( e ) Frequency of CD8 + T cells in subcutaneous PDAC tumors. DC, dendritic cells; MDSC, myeloid-derived suppressor cells; NK, natural killer cells; NKT, natural killer T cells; T reg , regulatory T cells. Data were analyzed 14 days post tumor implantation or at the time points indicated. Horizontal bars mark medians, error bars mark s.e.m. n indicates individual mice analyzed separately in at least two independent experiments with n≥2/group. P values determined by one-way ANOVA ( d ).

    Article Snippet: Finally, sections were incubated with anti-CD45 antibodies (DAKO, clone 2B11 + PD7/26) for 1 hour, followed by detection with Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide Alexa Fluor 647 (Invitrogen).

    Techniques: Derivative Assay, RNA Sequencing Assay, Purification, Mouse Assay, Expressing, Tumor Implantation

    Host-derived IL33 activates pancreatic ILC2s. ( a . ( b ) Representative IL33 immunohistochemistry (IHC) of IL33 Low and IL33 High human (tissue microarray, n=96) and mouse PDAC (n=3/group). ( c ) Frequency of human PDAC patients demonstrating IL33 positivity by IHC in a human PDAC tumor microarray. ( d ) Multiplexed immunofluorescence for IL33, ductal marker CK19, and myeloid markers CD11b, and Iba in mouse PDAC ( top ). Arrows, IL33-expressing cells. IL33 mean fluorescence intensity (MFI) in non-immune (CD45 − ), immune (CD45 + ), macrophage (TAM), and monocytic and granulocytic myeloid-derived suppressor cell (M-MDSC and G-MDSC) populations in tumors of IL33 Cit reporter PDAC mice ( bottom ). ( e ) Representative IL33 protein expression by IHC in orthotopic PDAC tumors in Il33 +/+ (WT) mice, and non-tumor-bearing pancreata in Il33 –/– mice (n=3/group). ( f ) ILC frequency (top) and cell number (bottom) in organs and draining lymph nodes (DLN) of Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( g ) Gating and frequency of IL4 and IL5 expression in intratumoral ILCs in Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( h ) ILC2 and ( i ) immune cell frequencies in orthotopic Rag2 –/– and Rag2 –/– γ c –/– PDAC mice with or without treatment with recombinant IL33 (rIL33). ( j ) Frequency of ST2 + tumor ILCs in mice with subcutaneous (SQ) and orthotopic PDAC. ( k ) Tumors in orthotopic and subcutaneous PDAC mice. ( l ) Tumor weight in Il33 +/+ and Il33 −/− littermate PDAC mice. ( m ) Experimental schema of bone-marrow chimeras to evaluate contribution of hematopoietic cell-derived IL33 to tumor control. ( n ) Hematopoietic cell reconstitution and ( o ) tumor weight in irradiated CD45.1 congenic mice reconstituted with either CD45.2 Il33 +/+ or CD45.2 Il33 −/− bone marrow. Data were collected at 14 ( a, d, f, g, j, o ), and 10 ( h, i ) days post tumor implantation. Horizontal bars mark medians. n indicates individual mice analyzed separately in at least two independent experiments with n≥2/group. P values were determined by one-way ANOVA ( a ) or two-tailed Mann-Whitney test ( d, f-h, j, l, o ).

    Journal: Nature

    Article Title: ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity

    doi: 10.1038/s41586-020-2015-4

    Figure Lengend Snippet: Host-derived IL33 activates pancreatic ILC2s. ( a . ( b ) Representative IL33 immunohistochemistry (IHC) of IL33 Low and IL33 High human (tissue microarray, n=96) and mouse PDAC (n=3/group). ( c ) Frequency of human PDAC patients demonstrating IL33 positivity by IHC in a human PDAC tumor microarray. ( d ) Multiplexed immunofluorescence for IL33, ductal marker CK19, and myeloid markers CD11b, and Iba in mouse PDAC ( top ). Arrows, IL33-expressing cells. IL33 mean fluorescence intensity (MFI) in non-immune (CD45 − ), immune (CD45 + ), macrophage (TAM), and monocytic and granulocytic myeloid-derived suppressor cell (M-MDSC and G-MDSC) populations in tumors of IL33 Cit reporter PDAC mice ( bottom ). ( e ) Representative IL33 protein expression by IHC in orthotopic PDAC tumors in Il33 +/+ (WT) mice, and non-tumor-bearing pancreata in Il33 –/– mice (n=3/group). ( f ) ILC frequency (top) and cell number (bottom) in organs and draining lymph nodes (DLN) of Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( g ) Gating and frequency of IL4 and IL5 expression in intratumoral ILCs in Il33 +/+ and Il33 −/− orthotopic PDAC mice. ( h ) ILC2 and ( i ) immune cell frequencies in orthotopic Rag2 –/– and Rag2 –/– γ c –/– PDAC mice with or without treatment with recombinant IL33 (rIL33). ( j ) Frequency of ST2 + tumor ILCs in mice with subcutaneous (SQ) and orthotopic PDAC. ( k ) Tumors in orthotopic and subcutaneous PDAC mice. ( l ) Tumor weight in Il33 +/+ and Il33 −/− littermate PDAC mice. ( m ) Experimental schema of bone-marrow chimeras to evaluate contribution of hematopoietic cell-derived IL33 to tumor control. ( n ) Hematopoietic cell reconstitution and ( o ) tumor weight in irradiated CD45.1 congenic mice reconstituted with either CD45.2 Il33 +/+ or CD45.2 Il33 −/− bone marrow. Data were collected at 14 ( a, d, f, g, j, o ), and 10 ( h, i ) days post tumor implantation. Horizontal bars mark medians. n indicates individual mice analyzed separately in at least two independent experiments with n≥2/group. P values were determined by one-way ANOVA ( a ) or two-tailed Mann-Whitney test ( d, f-h, j, l, o ).

    Article Snippet: Finally, sections were incubated with anti-CD45 antibodies (DAKO, clone 2B11 + PD7/26) for 1 hour, followed by detection with Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide Alexa Fluor 647 (Invitrogen).

    Techniques: Derivative Assay, Immunohistochemistry, Microarray, Immunofluorescence, Marker, Expressing, Fluorescence, Mouse Assay, Recombinant, Irradiation, Tumor Implantation, Two Tailed Test, MANN-WHITNEY

    ILC2s induce antigen-specific CD8+ T cell priming. ( a ) Gating and frequency of intratumoral ILC2s in ILC2-intact mice (diphtheria toxin [DT]-treated Icos +/+ ; CD4 Cre/+ ) and ILC2-depleted mice (DT-treated Icosfl . DTR/+ ; CD4 Cre/+ ). ( b ) Gating and frequency of OVA-specific CD8 + T cells in spleens from ILC-intact and ILC-depleted mice. OVA-specific T cells were detected as SIINFEKL-tetramer + cells. ( c ) Gating and frequency of central memory CD8 + T (T CM ) cells (CD45 + CD3 + CD8 + CD44 + CD62L + ) in tumor draining lymph nodes and spleens in ILC-intact and ILC-depleted mice. ( d ) ST2 expression on CD45 + CD3 + CD8 + T cells after tumor implantation in PDAC mice. Data were collected at 14 days post tumor implantation or at the time points indicated. DLN, draining lymph node; MFI, mean fluorescence intensity. Horizontal bars mark medians; error bars mark s.e.m. n indicates individual mice analyzed separately in at least two independent experiments with n≥2/group. P values determined by two-tailed Mann-Whitney test ( a-c ) and two-way ANOVA with Tukey’s multiple comparison post-test ( d , indicating comparison of tumor ILCs to all other groups).

    Journal: Nature

    Article Title: ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity

    doi: 10.1038/s41586-020-2015-4

    Figure Lengend Snippet: ILC2s induce antigen-specific CD8+ T cell priming. ( a ) Gating and frequency of intratumoral ILC2s in ILC2-intact mice (diphtheria toxin [DT]-treated Icos +/+ ; CD4 Cre/+ ) and ILC2-depleted mice (DT-treated Icosfl . DTR/+ ; CD4 Cre/+ ). ( b ) Gating and frequency of OVA-specific CD8 + T cells in spleens from ILC-intact and ILC-depleted mice. OVA-specific T cells were detected as SIINFEKL-tetramer + cells. ( c ) Gating and frequency of central memory CD8 + T (T CM ) cells (CD45 + CD3 + CD8 + CD44 + CD62L + ) in tumor draining lymph nodes and spleens in ILC-intact and ILC-depleted mice. ( d ) ST2 expression on CD45 + CD3 + CD8 + T cells after tumor implantation in PDAC mice. Data were collected at 14 days post tumor implantation or at the time points indicated. DLN, draining lymph node; MFI, mean fluorescence intensity. Horizontal bars mark medians; error bars mark s.e.m. n indicates individual mice analyzed separately in at least two independent experiments with n≥2/group. P values determined by two-tailed Mann-Whitney test ( a-c ) and two-way ANOVA with Tukey’s multiple comparison post-test ( d , indicating comparison of tumor ILCs to all other groups).

    Article Snippet: Finally, sections were incubated with anti-CD45 antibodies (DAKO, clone 2B11 + PD7/26) for 1 hour, followed by detection with Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide Alexa Fluor 647 (Invitrogen).

    Techniques: Mouse Assay, Expressing, Tumor Implantation, Fluorescence, Two Tailed Test, MANN-WHITNEY

    PD-1 blockade activates TILC2s. ( a ) Tumor volume and survival, ( b ) gating, frequency and number and ( c ) scRNA-seq (n=7,022 ILC2 single cells) in treated PDAC mice in a nonlinear representation of the top 15 principal components. Cells are colored by cluster ( left ) or treatment and tissue ( right ). In ( a, b ), n=n in left, right graphs respectively. ( d ) Tumor volume in wild-type (WT) or ILC2 deficient PDAC mice treated with αPD-1 + rIL33. ( e ) TILC2s were sort-purified from rIL33-treated WT or Pdcd1 −/− PDAC mice, transferred into ILC2-deficient PDAC recipients, and tumor volumes measured. ( f-h ) TILC2s were sort-purified from rIL33-treated PDAC CD45.1 donor mice, transferred into ILC2-deficient CD45.2 PDAC recipient mice, and treated with αPD-1 post cell transfer. Tumor volume and tumor weight ( f ), frequency of CD45.1 and CD45.2 cells ( g ), and frequency of T cells ( h ) (TILC2s- : all groups, n=8; TILC2+ : spleen, n=9; DLN, n=7; tumor, n=7) in recipient mice 10 weeks post cell transfer. Frequencies in g = percentage of live donor- or recipient-derived immune cells. ( i ) Tumor volume (vehicle, n=13; other groups, n=10) and survival (vehicle and αPD-1, n=15; rIL33, n=24; rIL33+αPD-1, n=26) of treated PDAC mice (KPC 52 cells). DLN, draining lymph node. Data were collected at 5 weeks ( b ), 10 days ( c ), and 6 weeks ( d ) post orthotopic tumor cell implantation. Horizontal bars mark medians, error bars mark s.e.m. Data are pooled from ≥2 independent experiments with n≥3/group; n and data points denote individual mice analyzed separately. Data for scRNA-seq represent pooled purified single cells from biological replicates (vehicle n=10, rIL33 n=5, αPD-1 + rIL33 n=5). P values were determined by two-way ANOVA with Tukey’s multiple comparison post ( a, d-f, i , tumor volume), two-tailed Mann-Whitney ( b, d, g , h ), and two-sided log-rank ( a , i , survival curves) tests.

    Journal: Nature

    Article Title: ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity

    doi: 10.1038/s41586-020-2015-4

    Figure Lengend Snippet: PD-1 blockade activates TILC2s. ( a ) Tumor volume and survival, ( b ) gating, frequency and number and ( c ) scRNA-seq (n=7,022 ILC2 single cells) in treated PDAC mice in a nonlinear representation of the top 15 principal components. Cells are colored by cluster ( left ) or treatment and tissue ( right ). In ( a, b ), n=n in left, right graphs respectively. ( d ) Tumor volume in wild-type (WT) or ILC2 deficient PDAC mice treated with αPD-1 + rIL33. ( e ) TILC2s were sort-purified from rIL33-treated WT or Pdcd1 −/− PDAC mice, transferred into ILC2-deficient PDAC recipients, and tumor volumes measured. ( f-h ) TILC2s were sort-purified from rIL33-treated PDAC CD45.1 donor mice, transferred into ILC2-deficient CD45.2 PDAC recipient mice, and treated with αPD-1 post cell transfer. Tumor volume and tumor weight ( f ), frequency of CD45.1 and CD45.2 cells ( g ), and frequency of T cells ( h ) (TILC2s- : all groups, n=8; TILC2+ : spleen, n=9; DLN, n=7; tumor, n=7) in recipient mice 10 weeks post cell transfer. Frequencies in g = percentage of live donor- or recipient-derived immune cells. ( i ) Tumor volume (vehicle, n=13; other groups, n=10) and survival (vehicle and αPD-1, n=15; rIL33, n=24; rIL33+αPD-1, n=26) of treated PDAC mice (KPC 52 cells). DLN, draining lymph node. Data were collected at 5 weeks ( b ), 10 days ( c ), and 6 weeks ( d ) post orthotopic tumor cell implantation. Horizontal bars mark medians, error bars mark s.e.m. Data are pooled from ≥2 independent experiments with n≥3/group; n and data points denote individual mice analyzed separately. Data for scRNA-seq represent pooled purified single cells from biological replicates (vehicle n=10, rIL33 n=5, αPD-1 + rIL33 n=5). P values were determined by two-way ANOVA with Tukey’s multiple comparison post ( a, d-f, i , tumor volume), two-tailed Mann-Whitney ( b, d, g , h ), and two-sided log-rank ( a , i , survival curves) tests.

    Article Snippet: Finally, sections were incubated with anti-CD45 antibodies (DAKO, clone 2B11 + PD7/26) for 1 hour, followed by detection with Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide Alexa Fluor 647 (Invitrogen).

    Techniques: Mouse Assay, Purification, Derivative Assay, Two Tailed Test, MANN-WHITNEY

    Activated tumor ILC2s express PD-1 and co-exist with PD-1 + T cells. Orthotopic PDAC mice (C57Bl/6 WT, Pdcd1 −/− ). Live, CD45 + , lineage − , CD90 + , CD25 + , ST2 + tumor ILC2s (TILC2s) were sort-purified to 98% purity at day 10 post-implantation. 5 × 10 5 TILC2s were immediately transferred to orthotopic PDAC tumor-bearing Il7r Cre/+ Rora fl/fl (ILC2-deficient) CD45.2 mice on days 7 and 14 post-tumor implantation via i.p. injection. Control mice received equivalent volumes of PBS via i.p. injections. ( a ) Representative plots for TILC2 sort-purification ( top ) and post-sort purity ( bottom ). ( b . ( c ) Survival and intratumoral CD8 + T cell frequency of orthotopic KPC 4662-GFP and KPC 52 PDAC tumors; horizontal bars in c mark medians. ( d ) Frequency of PD-1 + ILC2s ( left ) and correlation with PD-1 + T cells ( right ) in human PDAC. ( e ) Linear regression analysis of IL33 and PD-1 mRNA in bulk tumor transcriptomes of short- and long-human PDAC survivors ( left ) and survival association of PD-1 + cells in tumor tissue microarrays of short-term and long-term PDAC survivors ( right ); high and low defined as higher or lower than the median for the cohort. ( f ) Model linking the IL33-TILC2 axis to T cell immunity in pancreatic cancer. ( g ; data represent pooled purified single cells from biological replicates of n=10 (vehicle). Data are representative of purity and PD-1 expression on sorted TILC2s in two independent experiments with n≥4/group ( a, b ). n and data points denote individual mice and patients analyzed separately. P values were determined by two-tailed Mann-Whitney ( c ), and two-sided log rank ( c, e , survival curves) tests, and linear regression ( d, e ). Figure 4e

    Journal: Nature

    Article Title: ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity

    doi: 10.1038/s41586-020-2015-4

    Figure Lengend Snippet: Activated tumor ILC2s express PD-1 and co-exist with PD-1 + T cells. Orthotopic PDAC mice (C57Bl/6 WT, Pdcd1 −/− ). Live, CD45 + , lineage − , CD90 + , CD25 + , ST2 + tumor ILC2s (TILC2s) were sort-purified to 98% purity at day 10 post-implantation. 5 × 10 5 TILC2s were immediately transferred to orthotopic PDAC tumor-bearing Il7r Cre/+ Rora fl/fl (ILC2-deficient) CD45.2 mice on days 7 and 14 post-tumor implantation via i.p. injection. Control mice received equivalent volumes of PBS via i.p. injections. ( a ) Representative plots for TILC2 sort-purification ( top ) and post-sort purity ( bottom ). ( b . ( c ) Survival and intratumoral CD8 + T cell frequency of orthotopic KPC 4662-GFP and KPC 52 PDAC tumors; horizontal bars in c mark medians. ( d ) Frequency of PD-1 + ILC2s ( left ) and correlation with PD-1 + T cells ( right ) in human PDAC. ( e ) Linear regression analysis of IL33 and PD-1 mRNA in bulk tumor transcriptomes of short- and long-human PDAC survivors ( left ) and survival association of PD-1 + cells in tumor tissue microarrays of short-term and long-term PDAC survivors ( right ); high and low defined as higher or lower than the median for the cohort. ( f ) Model linking the IL33-TILC2 axis to T cell immunity in pancreatic cancer. ( g ; data represent pooled purified single cells from biological replicates of n=10 (vehicle). Data are representative of purity and PD-1 expression on sorted TILC2s in two independent experiments with n≥4/group ( a, b ). n and data points denote individual mice and patients analyzed separately. P values were determined by two-tailed Mann-Whitney ( c ), and two-sided log rank ( c, e , survival curves) tests, and linear regression ( d, e ). Figure 4e

    Article Snippet: Finally, sections were incubated with anti-CD45 antibodies (DAKO, clone 2B11 + PD7/26) for 1 hour, followed by detection with Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide Alexa Fluor 647 (Invitrogen).

    Techniques: Mouse Assay, Purification, Tumor Implantation, Injection, Expressing, Two Tailed Test, MANN-WHITNEY