anti cd4 antibody in pbs  (Thermo Fisher)

Journal: Journal of Neuro-Oncology

Article Title: The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

doi: 10.1007/s11060-024-04766-4

Figure Lengend Snippet: High CD25 expression correlates with robust FOXP3 expression in CD4+ positive canine T lymphocytes A ) Representative flow cytometric plot illustrating a low percentage (26.1%) of FOXP3+ cells when gating includes all CD25+CD4+ cells. B ) Representative flow cytometric plot illustrating a positive correlation between FOXP3 and CD25 expression in CD4+ canine T lymphocytes. In this example, FOXP3 expression was identified in 70.7% of cells with the highest CD25 expression (1.83%; red box). C ) Scatter dot plot demonstrating the positive correlation between FOXP3 and CD25 expression. D ) FOXP3 geometric mean fluorescence intensity and E ) mRNA expression was significantly increased in CD4+ CD25high T lymphocytes compared to CD4+ CD25low and CD8+ T lymphocytes. F ) Positive immunoreactivity for FOXP3 was observed co-localizing with nuclei of CD4+CD25high T lymphocytes. Three biological replicates were tested ( n =3 dogs) with three technical replicates per sample. Comparisons are based on one-way ANOVA with Tukey’s multiple comparisons test and unpaired two-tailed student’s t-test, where bars represent the group mean with the standard error of the mean (SEM). * p <0.05

Article Snippet: Freshly isolated PBMCs were stained with mixtures of Pacific Blue (PB) conjugated anti-dog CD4 (clone YKIX302.9; Thermo Fischer Scientific) and Phycoerythrin (PE) conjugated anti-dog CD25 (clone P4A10; Thermo Fischer Scientific).

Techniques: Expressing, Fluorescence, Two Tailed Test

Journal: Journal of Neuro-Oncology

Article Title: The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

doi: 10.1007/s11060-024-04766-4

Figure Lengend Snippet: Canine Tregs has the highest expression of CCR4 amongst T-cells. A ) Representative flow cytometric plot illustrating the percentage of CCR4+ cells within CD4+CD25+ and CD8+ canine T lymphocytes. In this example, CCR4 expression was identified in 28.5% of CD4+ CD25high cells and 8.6% of CD4+ CD25low cells. The expression of CCR4 in CD8+ T lymphocytes was minimal, 1.71%. B ) The percentage of CCR4+ cells identified via flow cytometry and C ) mRNA expression was significantly increased in CD4+CD25high T lymphocytes (2.43-fold increase) compared to CD4+CD25low and CD8+ T lymphocytes. Three biological replicates were tested ( n =3 dogs) with three technical replicates per sample. Comparisons are based on one-way ANOVA with Tukey’s multiple comparisons test and unpaired two-tailed student’s t-test, where bars represent the group mean with standard error of the mean (SEM). * p <0.05

Article Snippet: Freshly isolated PBMCs were stained with mixtures of Pacific Blue (PB) conjugated anti-dog CD4 (clone YKIX302.9; Thermo Fischer Scientific) and Phycoerythrin (PE) conjugated anti-dog CD25 (clone P4A10; Thermo Fischer Scientific).

Techniques: Expressing, Flow Cytometry, Two Tailed Test

Journal: Journal of Neuro-Oncology

Article Title: The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

doi: 10.1007/s11060-024-04766-4

Figure Lengend Snippet: The CCL2-CCR4 axis induces chemotaxis in canine Tregs. A ) Acutely isolated canine Tregs demonstrated robust chemotaxis toward human recombinant CCL2 (rhCCL2; 0.5 ug/ml) relative to media alone ( p =0.0007). This effect was normalized by adding anti-CCL2 antibody (5.0 ug/ml; p =0.15). B ) Chemotaxis of CD4+ helper T-cells was not affected by rhCCL2 ( p =0.2) or anti-CCL2 antibody ( p =0.4). C ) Chemotaxis of acutely isolated canine Tregs (CD4+CD25high) increased toward GSC0514 derived supernatant ( p <0.0001) and E ) J3T Bg derived supernatant ( p <0.0001). This effect was mitigated with the addition of anti CCL2 antibody (GSC0514, p =0.006; J3T Bg, p =0.0001) and abolished following Treg pre-treatment with CCR4 inhibitor (GSC0514 p <0.0001; J3T Bg p <0.0001) and a combination of anti-CCL2 and CCR4 inhibition (GSC0514 p <0.0001; J3T Bg p <0.0001). Migration of CD4+ helper T-cells was not affected by D ) GSC0514 derived supernatant ( p =0.06) nor perturbation of CCL2 ( p =0.8), CCR4 ( p =0.8) vs. combination of the two signaling ( p =0.09). Chemotaxis of CD4+ helper T-cells towards J3T Bg supernatant was induced, F ) ( p =0.02), and this effect was mitigated by anti-CCL2 antibody ( p =0.016); CCR4 inhibitor ( p =0.03) and combination of both ( p =0.001). Two independent experiments with technical duplicates per each condition were performed. Comparisons are based on one-way ANOVA with Tukey’s multiple comparisons test; bars represent the group mean with the standard error of the mean (SEM). **** p <0.0001

Article Snippet: Freshly isolated PBMCs were stained with mixtures of Pacific Blue (PB) conjugated anti-dog CD4 (clone YKIX302.9; Thermo Fischer Scientific) and Phycoerythrin (PE) conjugated anti-dog CD25 (clone P4A10; Thermo Fischer Scientific).

Techniques: Chemotaxis Assay, Isolation, Recombinant, Derivative Assay, Inhibition, Migration

Journal: Journal of Neuro-Oncology

Article Title: The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

doi: 10.1007/s11060-024-04766-4

Figure Lengend Snippet: Canine glioma CCL2 mRNA expression increases when exposed to Tregs but not conventional T-cells. mRNA levels of CCL2 were increased in A ) GSC0514, 0.95 ± 0.17- fold increased, p <0.001; B ) J3T Bg, 1.02 ± 0.33- fold increased, p <0.05; C ) GSC0111, 0.55 ± 0.11-fold increase, p <0.1; D ) G06A, 0.56 ± 0.19-fold increase, p <0.05, co-cultured with CD4+CD25high T lymphocytes relative to GSC0514, J3T Bg, GSC0111 and G06A cell lines without exposure to CD4+CD25high. mRNA levels of CCL2 were not significantly different when glioma cells were co-cultured with conventional T-cells. A ) 0.08 ± 0.05- fold decrease in GSC0514, p =0.8 B ) 0.30 ± 0.15 - fold increase in J3T-Bg, p =0.6 C ) 0.47 ± 0.29- fold decrease in GSC1110, p =0.2 and D ) 0.02 ± 0.1- fold decrease in G06A cell line, p =0.9. All reactions were run as 20μl triplicates per each condition. Comparisons are based on one-way ANOVA with Tukey’s multiple comparisons test; bars represent the group mean with the standard error of the mean (SEM). **** p <0.0001

Article Snippet: Freshly isolated PBMCs were stained with mixtures of Pacific Blue (PB) conjugated anti-dog CD4 (clone YKIX302.9; Thermo Fischer Scientific) and Phycoerythrin (PE) conjugated anti-dog CD25 (clone P4A10; Thermo Fischer Scientific).

Techniques: Expressing, Cell Culture

Journal: bioRxiv

Article Title: Massive detection of cryptic recessive genetic defects in cattle mining millions of life histories

doi: 10.1101/2023.09.22.558782

Figure Lengend Snippet: a top) Modeling of the 3D structure of the ITGα4β7 heterodimer using crystallography in humans, and evaluation of the consequences of glycine to serine substitution at amino acid position 283 in humans (375 in bovine). The purple dashed lines illustrate the clash with a predicted 0.8 kcal/mol reduction in binding affinity (see Methods for further details). a, down) Weblogo representation of multiple protein alignments showing perfect conservation of the mutated glycine residue among 128 vertebrate ITGB7 orthologs (Supplementary Table 19). b) Schematic representation of the ITGα4β7-mediated transmigration of T lymphocytes (T cells) from blood vessels to the lamina propria of the jejunum, and details of the sampling area for subsequent analyses. c) Dosage of α4β7pos CD45ROpos memory CD4 T cells in the lamina propria of the jejunum and of immune cells in the circulating blood of homozygous wild-type (0) and mutant (2) heifers (Supplementary Tables 20,21). ***: Student t test p-value ≤0.001. d) Pictures of 247-day-old wild-type and 245-day-old homozygous mutant heifers, illustrating growth retardation in the latter; and analysis of average daily gain in 13 cases and 13 matched control heifers aged six months to two years (Supplementary Table 22). e) Origin of H5P25 haplotype and ITGB7 mutation (see Supplementary Table 23 for details). f) Proportion of animals that died, were slaughtered, or are still alive at 2 years of age for each combination of ITGB7 variant genotype and H5P25 status. Different symbols indicate significant differences (Fisher p-value ≤0.01) between genotype-haplotype combinations in the proportions of animals within each of the three categories (Supplementary Table 24). g) Analysis of age at first AI for the three genotypes at the ITGB7 variant (Supplementary Table 25). ***: Student t test p-value ≤0.001. Effects of heterozygosity and homozygosity at the ITGB7 variant on 14 recorded traits (Supplementary Table 26). Only significant effects are shown (Benjamini-Hochberg adjusted Student’s t-test p-value ≤ 0.01). i) Trimester repartition of the proportion of females that died or were slaughtered before reaching six years of age by genotype group at the ITGB7 substitution (Supplementary Table 27).

Article Snippet: The antibodies used were: CD4-PB (clone CC8, Biorad, Hercules, U.S), CD45-FITC (clone 1.11.32, Biorad), CD45R0-A647 (clone IL-A116, Biorad), Beta7-RPE (clone FIB27, Biolegend, San Diego, U.S.), Alpha4-PECy7 (clone 9F10, Biolegend).

Techniques: Binding Assay, Residue, Transmigration Assay, Sampling, Mutagenesis, Variant Assay