antibodies anti-dog cd4 pe-cy7-conjugate clone ykix302.9 25-5040-42  (Thermo Fisher)

Journal: Cell Reports Medicine

Article Title: Cross-organ hierarchy of HLA molecular mismatches in donor-specific antibody development in solid organ transplantations

doi: 10.1016/j.xcrm.2025.102153

Figure Lengend Snippet: Examination of eplet mismatch immunogenicity in mixed lymphocyte reactions The association between the eplet risk score (ERS) and the activation status of responder CD4 + T cells was analyzed. Representative scattergrams are shown in (A), and aggregated scatterplots are shown in (B). Each stimulator-responder pair was analyzed with 3–6 biological replicates. The effect of an anti-55PP antibody (Ab) or an anti-45EV Ab on the percentage of CFSE low CD4 + T cells in the MLR was assessed using a stimulator-responder pair (pair #4, see ). Representative scattergrams are shown in (C), and aggregated data are shown in (D) and (E). Each antibody concentration group was analyzed with 4–12 biological replicates. ∗ p < 0.05. p values were determined using the Wilcoxon rank-sum test, which compared the value at 0 ng/mL Ab with those at 1 × 10 2 and 1 × 10 3 ng/mL Ab. EMn, the number of eplet mismatches; FSC, forward scatter; stim, stimulator. In the scatterplot shown in (B), large dots represent the median value of each stimulator-responder pair, small dots represent values obtained from each individual experiment, vertical lines represent ranges, and the gray area represents 95% confidence interval (CI) of the regression line. In the bar charts shown in (D) and (E), the top of the bar represents the median value, whereas the points represent values from individual experiments.

Article Snippet: Anti-CD4 PE-Cy7 antibody (clone SK3) , Becton, Dickinson and Company , Cat#348789; RRID: AB_400379.

Techniques: Immunopeptidomics, Activation Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: The transcription factor Nrf2 links Th2-mediated experimental allergy to food preservatives

doi: 10.3389/fimmu.2024.1476480

Figure Lengend Snippet: An increase in plasma levels of mast cell protease-1 and Th2 cell counts in tBHQ-fed mice occurs concurrently with worsened anaphylaxis. (A) Plasma levels of murine mast cell protease (mMCP)-1 1 h after oral challenge as determined by ELISA. (B) Pearson analysis of correlation between mMCP-1 plasma concentrations and body temperature 10 min post-challenge. Data are presented as mean ± SE. *Student’s t test: p < 0.05 as compared to CTL diet group at 1 h post-challenge. (n=6-7). (C) The percent degranulation of mast cells in duodenum as determined by toluidine blue staining. Toluidine blue positive metachromatic mast cells were counted in the tissue sections from OVA-sensitized mice on the tBHQ diet, control diet and saline-sensitized mice. In general, 2 sections per tissue per animal were examined under the microscope. Numbers of mast cells (intact and degranulated) in 40 high power fields (HPF) were counted and percent degranulation was calculated as the degranulation cell count over the total mast cell count. (n=3 mice/each group) *Student’s t test: p < 0.05 as compared to control diet (CTL, n=3). (D) Representative dot plots for Th2 cells (CD4 + GATA-3 + IL-4 + ) including percentages within gates for CTL diet versus tBHQ. (E) The percentage and (F) the absolute counts of Th2 cells among splenic CD4 + T cells from mice on either a CTL or tBHQ diet. Data are presented as mean ± SE. *Student’s t test: p < 0.05 as compared to control diet (CTL, n=4).

Article Snippet: Spleen cells (2 × 10 6 cells/ml) in DMEM (Genesee Scientific, El Cajon, CA) containing 10% fetal bovine serum (Gibco), 25 mM HEPES (Sigma), 10 mM nonessential amino acids (Sigma), 100 u/mL penicillin-streptomycin solution (Sigma), and 55μM 2-mercaptoethanol (Invitrogen) were first stimulated with anti-CD3 (1.5 μg/ml) (eBioscience Inc., San Diego, CA)/anti-CD28 (1.5 μg/ml) (Invitrogen) and crosslinker AffiniPure F(ab’) 2 Fragment Goat Anti-Syrian Hamster IgG(H+L) (1.5 μg/ml) (Jackson ImmunoResearch, West Grove, PA, USA) for 72 hours in the presence of 2 mM monensin (Biolegend, San Diego, CA), and then stained with viability dye (eBioscience Inc., San Diego, CA) and antibodies to cell surface markers, CD3-AF488 (Biolegend, San Diego, CA), CD4 + -PE/Cy7 (eBioscience Inc., San Diego, CA) and CD8a-PerCP/Cy5.5 (Biolegend, San Diego, CA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining, Control, Saline, Microscopy, Cell Counting

Journal: Frontiers in Immunology

Article Title: The transcription factor Nrf2 links Th2-mediated experimental allergy to food preservatives

doi: 10.3389/fimmu.2024.1476480

Figure Lengend Snippet: Mice on the tBHQ diet have a potentiated ex vivo recall response to OVA. Splenocytes were isolated from mice on either a control (CTL) or tBHQ diet following sensitization and oral challenge to OVA. The isolated splenocytes were cultured in the presence and absence of OVA (1mg/well) for 96 h in an ex vivo recall response. Quantification of (A) IL-4, (B) IL-5 or (C) IL-13 in cell supernatants by ELISA. (D) The percentage of CD4 + GATA-3 + IL-4 + cells (Th2 cells) among total splenocyte population. Data are presented as mean ± SE. *Student’s t test: p < 0.05 as compared to the CTL (n=4).

Article Snippet: Spleen cells (2 × 10 6 cells/ml) in DMEM (Genesee Scientific, El Cajon, CA) containing 10% fetal bovine serum (Gibco), 25 mM HEPES (Sigma), 10 mM nonessential amino acids (Sigma), 100 u/mL penicillin-streptomycin solution (Sigma), and 55μM 2-mercaptoethanol (Invitrogen) were first stimulated with anti-CD3 (1.5 μg/ml) (eBioscience Inc., San Diego, CA)/anti-CD28 (1.5 μg/ml) (Invitrogen) and crosslinker AffiniPure F(ab’) 2 Fragment Goat Anti-Syrian Hamster IgG(H+L) (1.5 μg/ml) (Jackson ImmunoResearch, West Grove, PA, USA) for 72 hours in the presence of 2 mM monensin (Biolegend, San Diego, CA), and then stained with viability dye (eBioscience Inc., San Diego, CA) and antibodies to cell surface markers, CD3-AF488 (Biolegend, San Diego, CA), CD4 + -PE/Cy7 (eBioscience Inc., San Diego, CA) and CD8a-PerCP/Cy5.5 (Biolegend, San Diego, CA).

Techniques: Ex Vivo, Isolation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: The transcription factor Nrf2 links Th2-mediated experimental allergy to food preservatives

doi: 10.3389/fimmu.2024.1476480

Figure Lengend Snippet: Sensitization and anaphylaxis to OVA is markedly diminished in SCID mice receiving Nrf2-deficient CD4 + T cells by adoptive transfer. SCID mice received wild-type or Nrf2-deficient CD4 + T cells via adoptive transfer (all animals received wild-type B cells) prior to OVA sensitization. Plasma levels of (A) : OVA-IgE (at 1/40 dilution) or (B) : OVA-IgG1 (at 1/2000 dilution) from individual animals were quantified by ELISA. *p < 0.05 as determined by two-way ANOVA with Bonferroni’s multiple comparisons test for post hoc analysis (n=7/WT on the control diet group, n=6/KO on the control diet group, n=7/WT on the diet with tBHQ group, n=7/KO on the diet with tBHQ). Data are presented as mean ± SE. (C-H) SCID mice receiving wild-type or Nrf2-deficient CD4 + T cells via adoptive transfer were challenged orally to OVA one week after the final sensitization. Rectal temperatures recorded before and at every 10 min after oral challenge in mice receiving (C) WT or (D) Nrf2-deficient CD4 + T cells. (E) Clinical scores of mice orally challenged with OVA (10 mg/mouse). (F) mMCP-1 plasma levels 1 h after oral challenge as determined by ELISA. The expression levels of (G) GATA3 and (H) IL-4 in CD4 + T cells as determined by mean fluorescence intensity by flow cytometry. Data are presented as mean ± SE. *p < 0.05 as determined by two-way ANOVA followed by Holm-Sidak or Bonferroni’s post hoc test (n=6-7). ND, Not detected. Statistical analysis for clinical scores between multiple groups was performed using a Kruskal-Wallis test with Dunn’s post hoc test.

Article Snippet: Spleen cells (2 × 10 6 cells/ml) in DMEM (Genesee Scientific, El Cajon, CA) containing 10% fetal bovine serum (Gibco), 25 mM HEPES (Sigma), 10 mM nonessential amino acids (Sigma), 100 u/mL penicillin-streptomycin solution (Sigma), and 55μM 2-mercaptoethanol (Invitrogen) were first stimulated with anti-CD3 (1.5 μg/ml) (eBioscience Inc., San Diego, CA)/anti-CD28 (1.5 μg/ml) (Invitrogen) and crosslinker AffiniPure F(ab’) 2 Fragment Goat Anti-Syrian Hamster IgG(H+L) (1.5 μg/ml) (Jackson ImmunoResearch, West Grove, PA, USA) for 72 hours in the presence of 2 mM monensin (Biolegend, San Diego, CA), and then stained with viability dye (eBioscience Inc., San Diego, CA) and antibodies to cell surface markers, CD3-AF488 (Biolegend, San Diego, CA), CD4 + -PE/Cy7 (eBioscience Inc., San Diego, CA) and CD8a-PerCP/Cy5.5 (Biolegend, San Diego, CA).

Techniques: Adoptive Transfer Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Control, Expressing, Fluorescence, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: The transcription factor Nrf2 links Th2-mediated experimental allergy to food preservatives

doi: 10.3389/fimmu.2024.1476480

Figure Lengend Snippet: Intrinsic role for Nrf2 in modulating food allergy response as determined by effects on gene expression. SCID mice received wild-type (n=3) or Nrf2-deficient CD4 + T cells (n=3) via adoptive transfer (all animals received wild-type B cells) prior to sensitization and oral challenge to OVA. Wild-type and Nrf2-null splenocytes were analyzed to identify DEGs by using RT 2 Profiler PCR Arrays Test. (A) Gene expression of representative DEGs were presented as fold induction as compared to Nrf2-null group. DEGs were determined by setting the threshold for fold change > 2 and P-values < 0.05. Data are presented as mean ± SE. *Student’s t test: p < 0.05 as compared to the Nrf2-null group. (B) Grouping of networks based on functionally enriched GO terms and pathways by using ClueGO, Cytoscape software. Pathway groups were generated based on Kappa Score = 0.5. (C) Percentage of genes associated with various pathways relative to the total number of associated genes as determined by KEGG analysis.

Article Snippet: Spleen cells (2 × 10 6 cells/ml) in DMEM (Genesee Scientific, El Cajon, CA) containing 10% fetal bovine serum (Gibco), 25 mM HEPES (Sigma), 10 mM nonessential amino acids (Sigma), 100 u/mL penicillin-streptomycin solution (Sigma), and 55μM 2-mercaptoethanol (Invitrogen) were first stimulated with anti-CD3 (1.5 μg/ml) (eBioscience Inc., San Diego, CA)/anti-CD28 (1.5 μg/ml) (Invitrogen) and crosslinker AffiniPure F(ab’) 2 Fragment Goat Anti-Syrian Hamster IgG(H+L) (1.5 μg/ml) (Jackson ImmunoResearch, West Grove, PA, USA) for 72 hours in the presence of 2 mM monensin (Biolegend, San Diego, CA), and then stained with viability dye (eBioscience Inc., San Diego, CA) and antibodies to cell surface markers, CD3-AF488 (Biolegend, San Diego, CA), CD4 + -PE/Cy7 (eBioscience Inc., San Diego, CA) and CD8a-PerCP/Cy5.5 (Biolegend, San Diego, CA).

Techniques: Gene Expression, Adoptive Transfer Assay, Software, Generated