Journal: Open Medicine

Article Title: Luteolin alleviates ulcerative colitis in rats via regulating immune response, oxidative stress, and metabolic profiling

doi: 10.1515/med-2023-0785

Figure Lengend Snippet: Effects of LUT on inflammation and immune response in UC rats. (a) Expression levels of TNF-α, CRP, IL-13, IL-33, and ST-2 were examined by ELISA. (b) Representative image of CD4+ T cell staining in the colon (100× magnification). (c) Representative image of CD8+ T cell staining (100× magnification). NC represents normal control. MOD represents model. MES represent mesalazine. LUT represents luteolin. There were 10, 8, 8, and 9 replicates in NC, MOD, MES, and LUT groups, respectively. ** P < 0.01 vs the NC group. # P < 0.05 and ## P < 0.01 vs the MOD group.

Article Snippet: The sections were blocked with 5% BSA and incubated with primary antibody against CD4 (1:2,000; Abcam, UK) and CD8 (1:2,000; Abcam, UK) overnight at 4°C.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Scientific Reports

Article Title: Cytomegalovirus infection exacerbates autoimmune mediated neuroinflammation

doi: 10.1038/s41598-017-00645-3

Figure Lengend Snippet: CMV infection expands CD4 + CD28 null T cells. Flow cytometry was performed to determine the percentage of CD4 + CD28 null T cells. CMV and EBV status and immunoglobulin titers were determined via ELFA. CD4 + CD28 null T cells in ( a ) CMV seropositive (n = 100) compared to seronegative (n = 127) MS patients and ( b ) CMV seropositive (n = 24) versus seronegative healthy controls (n = 39). ( c ) Correlation of CMV IgG levels with the percentage of CD4 + CD28 null T cells in MS patients and HC (n = 140). ( d ) Correlation of EBV EBNA IgG titers in 155 MS patients. ( e ) Repeated stimulation of PBMCs from HC (n = 12) and MS patients (n = 8) with CMV pp65 ( e ) or IL-2 ( f ) in vitro , after which the number of CD4 + CD28 null T cells was determined at different time points. ( g ) Flow cytometry of splenocytes of MCMV infected mice on day 0, day 8 and day 250 post infection (n = 5/time point). ( h ) Splenocytes from MCMV-infected WT (n = 5) and CD80/86 −/− (n = 4) mice were analysed for the percentage of CD4 + CD28 null T cells at day 250 post infection. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Sections were fixed, blocked and incubated with antibodies against CD4 (1/100, BD Biosciences, 553043) and granzyme B (1/100, Abcam, Ab4059).

Techniques: Infection, Flow Cytometry, In Vitro

Journal: Scientific Reports

Article Title: Cytomegalovirus infection exacerbates autoimmune mediated neuroinflammation

doi: 10.1038/s41598-017-00645-3

Figure Lengend Snippet: CD4 + CD28 null T cells are increased in EAE mice, as a result of auto-antigenic stimulation. ( a ) The phenotype of blood-derived mouse CD4 + CD28 null T cells was measured via flow cytometry. ( b ) After induction, EAE mice (n = 15) and CFA control mice (n = 10) were scored for maximum 90 days according to their disability. ( c ) Blood was collected at different time point s , to determine the number of CD4 + CD28 null T cells via flow cytometry. ( d ) Correlation between CD4 + CD28 null T cells and EAE score. ( e ) Historical human MBP specific T cell clones repeatedly stimulated with MBP/PHA (n = 8) were thawed and analysed for the number of CD4 + CD28 null T cells via flow cytometry. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Sections were fixed, blocked and incubated with antibodies against CD4 (1/100, BD Biosciences, 553043) and granzyme B (1/100, Abcam, Ab4059).

Techniques: Derivative Assay, Flow Cytometry, Clone Assay

Journal: Scientific Reports

Article Title: Cytomegalovirus infection exacerbates autoimmune mediated neuroinflammation

doi: 10.1038/s41598-017-00645-3

Figure Lengend Snippet: MCMV infected mice with EAE have a worse disease course. Mice were infected with MCMV and after 8 days EAE was induced. ( a ) Daily scoring of the CMV, EAE and MCMV infected EAE groups (n = 8/group). ( b ) Splenocytes were isolated at day 0 (baseline) and day 30 and CD4 + CD28 null T cells were measured via flow cytometry. ( c ) After stimulation of splenocytes with a MOG 35–55 peptide, the IFNγ producing CD4 + T cells were measured via flow cytometry and normalized to the non-peptide control. The MOG response significantly correlated with the percentage of CD4 + CD28 null T cells in the spleen. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Sections were fixed, blocked and incubated with antibodies against CD4 (1/100, BD Biosciences, 553043) and granzyme B (1/100, Abcam, Ab4059).

Techniques: Infection, Isolation, Flow Cytometry

Journal: Scientific Reports

Article Title: Cytomegalovirus infection exacerbates autoimmune mediated neuroinflammation

doi: 10.1038/s41598-017-00645-3

Figure Lengend Snippet: CMV infection increases EAE lesion size. ( a ) Representative staining of MBP in the CMV control group (left), the EAE control group (middle) and the CMV infected EAE animals (right). ( b ) The amount of demyelination within the CMV and EAE control groups and the CMV infected EAE group, calculated via dividing the demyelinated area (=loss of MBP) in the white matter of the spinal cord, over the total white matter area for each section. ( c ) Correlation between the amount of demyelination for each animal and the percentage of peripheral CD4 + CD28 null T cells. ( d ) Single and double staining of CD4 and Granzyme B, which points to the presence of CD4 + CD28 null T cells, in the spinal cord of EAE mice. *p < 0.05, ****p < 0.0001.

Article Snippet: Sections were fixed, blocked and incubated with antibodies against CD4 (1/100, BD Biosciences, 553043) and granzyme B (1/100, Abcam, Ab4059).

Techniques: Infection, Staining, Double Staining

Journal: Scientific Reports

Article Title: Cytomegalovirus infection exacerbates autoimmune mediated neuroinflammation

doi: 10.1038/s41598-017-00645-3

Figure Lengend Snippet: Study subjects for CD4 + CD28 null T cell analysis.

Article Snippet: Sections were fixed, blocked and incubated with antibodies against CD4 (1/100, BD Biosciences, 553043) and granzyme B (1/100, Abcam, Ab4059).

Techniques:

Journal: Biomolecules

Article Title: Clinical Significance and Immune Infiltration Analyses of the Cuproptosis-Related Human Copper Proteome in Gastric Cancer

doi: 10.3390/biom12101459

Figure Lengend Snippet: Immune infiltration analyses. ( A – C ) Detection of infiltrated macrophages, CD8+ T cells and CD4+ T cells in 4 responders/CBP_low and 4 non-responder/CBP_high to immunotherapy in the PKUCH cohort. ( D – F ) The M1/M2 ratio, CD8+ T cell proportion and CD4+ T cell proportion in 4 responders/CBP_low and 4 non-responders/CBP_high to immunotherapy. ( G – I ) Kaplan–Meier curves for the OS of GC patients in different groups classified by the median of CD68, CD8 and CD4 levels from a PKUCH cohort.

Article Snippet: FFPE tissue slides were first deparaffinized in a BOND RX system (Leica Biosystems) and then incubated sequentially with primary antibodies targeting CD4 (1:100: Abcam, ab133616), CD8 (1:200, Abcam, ab178089), CD163 (1:500, Abcam, ab182422), CD68 (1:1000, Abcam, ab213363), PD-1 (1:200, CST, D4W2J, 86163S), PD-L1 (1:400, CST, E1L3N, 13684S) and CD20 (1:1, Dako, L26, IR604).

Techniques:

Journal: Nature communications

Article Title: Hemogenic endocardium contributes to transient definitive hematopoiesis

doi: 10.1038/ncomms2569

Figure Lengend Snippet: Immunohistochemistry for CD41 (blue) and CD31 (red) in outflow tract (OFT) and atria at E9.5. CD41 + endocardial cells are clustered in the outflow cushion and the dorsal wall of the atria, and also scattered in the entire circumference (arrows). Scale bar = 100µm (lower magnification), 50µm (higher magnification). Immunofluorescent staining for CD41 (red), CD31 (green) and DAPI (blue) in left atrium at E9.5. Arrows indicate scattered CD31 + endocardial cells co-expressing CD41. Scale bar = 20µm. Electron microscopic analysis of E10.0–10.5 atrium. Bottom panels show higher magnification of the broken-lined area in upper panels. Left panels indicate regular flat endocardial cells. Center panels are representative images of endocardial cells rounded up. Endothelial cells occasionally protrude into the cardiac lumen (right panels). The bottom panels are higher magnification of adherens junctions in the dotted area in the middle panels (arrows). Scale bar = 2µm (upper panels), 1µm (middle panels) and 100nm (bottom panels). Yellow = endocardial layer. Red = myocardial layer.

Article Snippet: The following primary antibodies were used: rat CD41 (1:50, BD PharMingen), Phospho-Histone 3 (1:200, Millipore), Ter119 (1:50 eBioscience), and PECAM-1/CD31 (1:200, BD PharMingen) were used on fixed frozen sections.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Nature communications

Article Title: Hemogenic endocardium contributes to transient definitive hematopoiesis

doi: 10.1038/ncomms2569

Figure Lengend Snippet: Immunofluorescent staining for Nkx2-5 (green) and CD31 (red) at E9.5. Clusters of Nkx2-5 + cells are found in the endocardium of outflow cushion, atrioventricular cushion, and the dorsal wall of the atria (white arrows). Note that myocardium shows stronger level of Nkx2-5 expression (white arrowheads). Endodermal cells are also positive for Nkx2-5 (left lower panel, open arrowhead). Scale bar = 50µm (lower magnification), 20µm (higher magnification). Heart section of Nkx2-5 IRES-Cre/+ ; R26 YFPR/+ embryo at E10.5 stained for YFP (green) and CD31 (red). Nkx2-5-derived endocardial cells (YFP/CD31 double positive) were clustered in the outflow cushion and the dorsal wall of the atrium, and also found scattered along the perimeter (arrows). This specific distribution pattern corresponds to that of CD41 + cells . Arrowheads indicate non-Nkx2-5-derived endocardial cells. Scale bar = 100µm (lower magnification), 20µm (higher magnification). Heart section of Nkx2-5 IRES-Cre/+ ; R26 YFPR/+ embryo at E10.5 stained for YFP (green) and CD41 (red). Nkx2-5 + cells give rise to CD41 + cells in the endocardium (YFP/CD41 double positive, arrows). Scale bar = 100µm (lower magnification), 20µm (higher magnification). Percentage of CD41 + cells in Nkx2-5-derived fraction of endocardium at E9.5. 48% of Nkx2-5-lineage labeled endocardial cells express CD41 whereas 4.4% are CD41-positive in non-Nkx2-5-derived endocardial cells. The graph represents the average of three independent experiments. Mean±SEM.

Article Snippet: The following primary antibodies were used: rat CD41 (1:50, BD PharMingen), Phospho-Histone 3 (1:200, Millipore), Ter119 (1:50 eBioscience), and PECAM-1/CD31 (1:200, BD PharMingen) were used on fixed frozen sections.

Techniques: Staining, Expressing, Derivative Assay, Labeling

Journal: Nature communications

Article Title: Hemogenic endocardium contributes to transient definitive hematopoiesis

doi: 10.1038/ncomms2569

Figure Lengend Snippet: Nkx2-5 mutant heart at E9.5 stained for CD41 (blue) and CD31 (red). CD41 + cells were hardly found in the endocardium of Nkx2-5 mutants. Note that Nkx2-5 mutant also develops known cardiac defects including a single ventricle with poorly developed trabeculae, open atrioventricular canal, and lack of epithelial-mesenchymal transformation in the endocardial cushion. Scale bar = 100µm. Nkx2-5-derived endocardial cells in Nkx2-5 hetero (left panels) and null (right panels) background. Nkx2-5-driving cells were identified in the cushion endocardium even in the absence of Nkx2-5. Blood colony formation assay from FACS-purified endocardial cells. No colonies were formed from Nkx2-5 mutant endocardial cells. Data represent average of three independent experiments. Comparison of the peripheral blood cells derived from Nkx2-5-expressing cells in Nkx2-5-null and its control backgrounds at E10.0. YFP-labeled red blood cells (Ter119 + ) were found less frequently (0.2%) in Nkx2-5 Cre/Cre ; R26 YFPR/+ embryos compared with control littermates (1.7%), suggesting that expression of Nkx2-5 is required for the hemogenic activity of the Nkx2.5 + endocardial/endothelial cells in vivo .

Article Snippet: The following primary antibodies were used: rat CD41 (1:50, BD PharMingen), Phospho-Histone 3 (1:200, Millipore), Ter119 (1:50 eBioscience), and PECAM-1/CD31 (1:200, BD PharMingen) were used on fixed frozen sections.

Techniques: Mutagenesis, Staining, Transformation Assay, Derivative Assay, Colony Assay, Purification, Expressing, Labeling, Activity Assay, In Vivo

Journal: International Journal of Molecular Sciences

Article Title: Th17 Activation and Th17/Treg Imbalance in Prolonged Anterior Intraocular Inflammation after Ocular Alkali Burn

doi: 10.3390/ijms23137075

Figure Lengend Snippet: Immunofluorescence examination of normal control and OAB-subjected eyes ( n = 6 for each time point). Immunofluorescent staining of CD4 (green) and IL-17A (red) in the normal control and OAB-subjected eyes on the 1st, 3rd, 7th, 14th, 21st, and 28th day post modeling (40X). Cell nuclei were stained with DAPI (blue). CD4 and IL-17A double positive cells were found around the iris from days 7 to 28. Scale bar: 20 μm.

Article Snippet: Sections were blocked with 5% bovine serum albumin and incubated with primary antibodies against CD4 (1:50, NBP1-19371, Novus Biologicals, Centennial, CO, USA) and IL-17A (1:50, sc-374218, Santa Cruz, CA, USA) at 4 °C overnight.

Techniques: Immunofluorescence, Control, Staining