cd4 fitc labeled antibody  (Bio-Rad)

Journal: International Journal of Molecular Sciences

Article Title: Enhanced ROS Production and Mitochondrial Metabolic Shifts in CD4 + T Cells of an Autoimmune Uveitis Model

doi: 10.3390/ijms252111513

Figure Lengend Snippet: CD4 + T cells from ERU cases produced higher levels of reactive oxygen species (ROS) after stimulation with pokeweed mitogen (PWM). Bar graphs display the factor of ROS production in CD4 + T cells from healthy horses (Ctr, gray, n = 6) and equine recurrent uveitis cases (ERU, red, n = 5) after 90 min of stimulation with PWM. The mean value is shown as a factor and the error bars correspond to the standard deviation. The factor of ROS production in control cells was determined at 3.6, while ERU cases exhibited a factor of 6.1. To compare the factor of ROS production between CD4 + T cells from ERU cases and healthy control cells, Student’s t -test was used (* p = 0.023).

Article Snippet: The purity of the CD4 + T cell fraction was tested via flow cytometry after staining of 2 × 10 5 cells of each fraction with 30 μL mouse anti-horse CD4 FITC labeled antibody (clone MCA1078F, Biorad, Feldkirchen, Germany, 1:10), which resulted in a mean purity of 98% ± 1.7% SD. ( ).

Techniques: Produced, Standard Deviation, Control

Journal: International Journal of Molecular Sciences

Article Title: Enhanced ROS Production and Mitochondrial Metabolic Shifts in CD4 + T Cells of an Autoimmune Uveitis Model

doi: 10.3390/ijms252111513

Figure Lengend Snippet: Lower dependency on and higher flexibility for mitochondrial oxidation of glucose in ERU cases. Bar graphs show the percentage of dependency on mitochondrial glucose oxidation in CD4 + T cells from healthy controls (gray, n = 6) and ERU cases (red, n = 5) as well as capacity and flexibility from healthy controls (gray, n = 5) and ERU cases (red, n = 5). The mean value is presented as a percentage, and the error bars correspond to the standard deviation. Both groups showed 100% capacity to use glucose for mitochondrial oxidation (ns = not significant), whereas CD4 + T cells from ERU cases were significantly (** p = 0.009) less dependent and exhibited significantly (* p = 0.012) greater flexibility to use glucose as a mitochondrial fuel compared to controls. Student’s t -test was used to test for statistical significance.

Article Snippet: The purity of the CD4 + T cell fraction was tested via flow cytometry after staining of 2 × 10 5 cells of each fraction with 30 μL mouse anti-horse CD4 FITC labeled antibody (clone MCA1078F, Biorad, Feldkirchen, Germany, 1:10), which resulted in a mean purity of 98% ± 1.7% SD. ( ).

Techniques: Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Enhanced ROS Production and Mitochondrial Metabolic Shifts in CD4 + T Cells of an Autoimmune Uveitis Model

doi: 10.3390/ijms252111513

Figure Lengend Snippet: Mitochondrial fuel utilization of glutamine did not show significant differences in CD4 + T cells from ERU cases. Bar graphs show the percentage of capacity and flexibility of mitochondrial glutamine oxidation in CD4 + T cells from healthy controls (gray, n = 6) and ERU cases (red, n = 5). Flexibility was calculated by subtracting the dependency from the capacity (flexibility% = capacity% − dependency%). The mean value is shown as a percentage and the error bars correspond to the standard deviation. Student’s t -test revealed no statistically significant (ns p = 0.066) differences in capacity or flexibility of substrate utilization of glutamine.

Article Snippet: The purity of the CD4 + T cell fraction was tested via flow cytometry after staining of 2 × 10 5 cells of each fraction with 30 μL mouse anti-horse CD4 FITC labeled antibody (clone MCA1078F, Biorad, Feldkirchen, Germany, 1:10), which resulted in a mean purity of 98% ± 1.7% SD. ( ).

Techniques: Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Enhanced ROS Production and Mitochondrial Metabolic Shifts in CD4 + T Cells of an Autoimmune Uveitis Model

doi: 10.3390/ijms252111513

Figure Lengend Snippet: CD4 + T cells from ERU cases showed greater capacity and flexibility to utilize long-chain fatty acids (LCFAs) for mitochondrial energy production. Bar graphs represent the percentage of capacity and flexibility for mitochondrial oxidation of LCFAs in CD4 + T cells from healthy controls (gray, n = 6) and ERU cases (red, n = 5). Flexibility was calculated by subtracting the dependency from the capacity (flexibility% = capacity% − dependency%). The mean value is shown as a percentage and the error bars correspond to the standard deviation. ERU cases showed significantly (* p = 0.041) higher capacity and flexibility to use LCFAs for mitochondrial oxidation. Student’s t-test was used to test for statistical significance.

Article Snippet: The purity of the CD4 + T cell fraction was tested via flow cytometry after staining of 2 × 10 5 cells of each fraction with 30 μL mouse anti-horse CD4 FITC labeled antibody (clone MCA1078F, Biorad, Feldkirchen, Germany, 1:10), which resulted in a mean purity of 98% ± 1.7% SD. ( ).

Techniques: Standard Deviation

Journal: Frontiers in Immunology

Article Title: Altered Metabolic Phenotype of Immune Cells in a Spontaneous Autoimmune Uveitis Model

doi: 10.3389/fimmu.2021.601619

Figure Lengend Snippet: Oxygen consumption rates (OCR) and OCR ratios by mitochondrial metabolism of PBMC, CD4 + T cells and CD4 - cells of healthy and ERU cases. Controls (n=16) are presented as grey lines whereas ERU cases (n=9) are illustrated as blue lines. OCR is shown by graph (A) and OCR ratios are illustrated by graphs (B–D) . First three measurements (dots) show basal respiration, followed by injections of oligomycin (after the third measurement point), FCCP (after the sixth measurement point) and Rot/AA (after the ninth measurement point). Data are shown as mean ± SEM. (A) Significantly higher basal oxygen consumption rates of equine PBMC (**p ≤ 0.01), CD4 + T cells (***p ≤ 0.001) and CD4 - cells (**p ≤ 0.01) of ERU cases compared to controls. (B) After normalization against the baseline mean, PBMC of ERU cases showed significantly lower OCR ratio after FCCP (***p ≤ 0.001) and Rot/AA (**p ≤ 0.01) injection compared to controls. (C) CD4 + T cells of ERU cases showed significantly higher OCR ratio (***p ≤ 0.001) when injected with oligomycin and Rot/AA. (D) CD4 - cells of horses with ERU had significantly increased OCR ratio (**p ≤ 0.01) after FCCP injection. No significant difference is represented by ns (ns p > 0.05).

Article Snippet: Purity of CD4 + fraction was tested via flow cytometry, by staining with 30 μl mouse anti horse CD4 FITC labelled antibody (clone MCA1078F, Biorad, Feldkirchen, Germany, 1:10) and 30 μl mouse anti horse CD8 FITC labelled antibody (clone MCA1080F, Biorad, Feldkirchen, Germany, 1:10) ( , ≥ 96% purity of the CD4 + fraction).

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: Altered Metabolic Phenotype of Immune Cells in a Spontaneous Autoimmune Uveitis Model

doi: 10.3389/fimmu.2021.601619

Figure Lengend Snippet: Extracellular acidification rate (ECAR) of PBMC, CD4 + T cells and CD4 - cells of healthy and ERU cases. Graphs display the extracellular acidification rate (ECAR) of cells of healthy (grey, n=14) and ERU diseased cases (blue, n=8). Graph (A) shows extracellular acidification rates, graphs (B–D) show ECAR ratios of healthy and ERU cases. First three measurements (dots) show basal respiration, followed by injections of oligomycin (after the third measurement point), FCCP (after the sixth measurement point) and Rot/AA (after the ninth measurement point). Data are shown as mean ± SEM. (A) PBMC and more specifically CD4 - cells of ERU cases showed significantly increased basal extracellular acidification (***p ≤ 0.001) compared to controls, whereas no significant difference (ns p > 0.05) between the basal extracellular acidification rate of CD4 + T cells of healthy and diseased horses was detectable. (B) ECAR ratio of PBMC of ERU cases decreased significantly after injection of the reagents of the mito stress test. (C) CD4 + T cells had a significantly lower ECAR ratio than controls after injection of oligomycin, FCCP and Rot/AA. (D) ECAR ratio of CD4 - cells was significantly lower in ERU cases than in controls after injection of the mito stress test reagents. Statistical significances are represented by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

Article Snippet: Purity of CD4 + fraction was tested via flow cytometry, by staining with 30 μl mouse anti horse CD4 FITC labelled antibody (clone MCA1078F, Biorad, Feldkirchen, Germany, 1:10) and 30 μl mouse anti horse CD8 FITC labelled antibody (clone MCA1080F, Biorad, Feldkirchen, Germany, 1:10) ( , ≥ 96% purity of the CD4 + fraction).

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: Altered Metabolic Phenotype of Immune Cells in a Spontaneous Autoimmune Uveitis Model

doi: 10.3389/fimmu.2021.601619

Figure Lengend Snippet: Glycolytic proton efflux (glycoPER) of PBMC, CD4 + T cells and CD4 - cells of healthy and ERU cases. Graph (A) shows the glycolytic proton efflux rates of controls (grey, n=8) and ERU cases (blue, n=10), graphs (B–D) represent glycoPER ratios, normalized against the baseline mean. First three measurements (dots) show basal respiration, followed by injections of Rot/AA (after the third measurement point) and 2-DG (after the sixth measurement point). Data are shown as mean ± SEM. (A) Significantly increased basal glycolysis (***p ≤ 0.001) of PBMC and more precisely CD4 - cells of ERU cases, whereas CD4 + T cells of horses with ERU showed significantly lower basal glycolysis (*p ≤ 0.05). (B) Significantly higher glycoPER ratio (*p ≤ 0.05) of ERU PBMC after Rot/AA injection. (C) Significantly increased glycoPER (*p ≤ 0.05) of CD4 + T cells of ERU cases after Rot/AA injection. (D) CD4 - cells showed no difference in glycolysis ratios (ns p > 0.05) after treatment with Rot/AA and 2-DG compared to controls.

Article Snippet: Purity of CD4 + fraction was tested via flow cytometry, by staining with 30 μl mouse anti horse CD4 FITC labelled antibody (clone MCA1078F, Biorad, Feldkirchen, Germany, 1:10) and 30 μl mouse anti horse CD8 FITC labelled antibody (clone MCA1080F, Biorad, Feldkirchen, Germany, 1:10) ( , ≥ 96% purity of the CD4 + fraction).

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: Altered Metabolic Phenotype of Immune Cells in a Spontaneous Autoimmune Uveitis Model

doi: 10.3389/fimmu.2021.601619

Figure Lengend Snippet: ATP5β in CD4 + T cells of healthy and ERU cases. (A) Western blot with CD4 + T cells of control and ERU horses (n=5 each) stained with anti ATP5β antibody, anti GAPDH antibody and anti β-actin antibody for normalization. A molecular weight marker is shown on the left, followed by a blank lane and a lane only incubated with the secondary antibody. (B) Statistical analysis of the volume of ATP5β lanes of control and ERU cases after normalization to the respective β-actin lane showed no significant difference (ns p > 0.05). Data are represented as mean ± SEM. The ERU bar (blue) was set accordingly to the control bar (grey), which was set as 1. (C) Representative immunocytochemically stained CD4 + T cell of a control (upper picture) and an ERU case (lower picture). Mitochondria were stained with MitoTracker Deep Red (red). Cell nuclei were counterstained with DAPI (blue). x1000 magnification.

Article Snippet: Purity of CD4 + fraction was tested via flow cytometry, by staining with 30 μl mouse anti horse CD4 FITC labelled antibody (clone MCA1078F, Biorad, Feldkirchen, Germany, 1:10) and 30 μl mouse anti horse CD8 FITC labelled antibody (clone MCA1080F, Biorad, Feldkirchen, Germany, 1:10) ( , ≥ 96% purity of the CD4 + fraction).

Techniques: Western Blot, Control, Staining, Molecular Weight, Marker, Incubation