Journal: Scientific Reports

Article Title: A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma

doi: 10.1038/s41598-017-09444-2

Figure Lengend Snippet: Enhancement of cytokine production and cell proliferation of dog peripheral blood mononuclear cells by c4G12 treatment. Dog peripheral blood mononuclear cells ( n = 7) were obtained from healthy beagle donors and stimulated by 5 μg/mL staphylococcal enterotoxin B in the presence or absence of 20 μg/mL c4G12. Dog IgG was used as a control antibody. For evaluation of cytokine production, the culture supernatant was harvested on day 3, and concentration of ( a ) IL-2 or ( b ) IFN-γ was measured by ELISA. To evaluate cell proliferation, nucleotide analogue 5-ethynyl-2′-deoxyuridine (EdU) was added to the medium on day 2, and cells were harvested after incubation for another 2 h. The lymphocyte population was gated by forward scatter and side scatter, and the incorporation of EdU in ( c ) CD4+ or ( d ) CD8+ cells was measured by a flow cytometer. Statistical analysis was performed with a Wilcoxon signed rank-sum test.

Article Snippet: For cytokine assays, the culture supernatant was harvested on day 3 and the concentrations of IL-2 and IFN-γ were measured by Duoset ELISA canine IL-2 and IFN-γ (R&D systems, Minneapolis, MN, USA), respectively, according to the manufacturer’s instructions.

Techniques: Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

Journal: bioRxiv

Article Title: IRAK4 autophosphorylation controls inflammatory signaling by activating IRAK oligomerization

doi: 10.1101/2023.12.21.572799

Figure Lengend Snippet: a) IL-2 release in WT and EL4-MyD88-GFP/IRAK4-mScarlet cells treated with DMSO or IRAK4 kinase inhibitor. IL-2 release was measured by ELISA 24 h after IL-1β stimulation. Values shown are the fold change in IL-2 release. Average values calculated from three independent experiments. Bars represent mean ± SEM. b) Treatment with the IRAK4 kinase inhibitor blocks pIRAK4 production after IL-1 stimulation. WT and EL4-MyD88-GFP/IRAK4-mScarlet cell lysates analyzed for pIRAK4 production by Western blot analysis. Cells treated with 20 µM of IRAK4 kinase inhibitor (or DMSO) for 4 h before being stimulated with 1 ng/ml of IL-1β for 30 mins in the presence of the inhibitor. c) TIRF images and kymograph analysis of DMSO control treated EL4-MyD88-GFP/IRAK4-mScarlet stimulated on IL-1 functionalized SLBs. Kymographs derived from red line overlaid TIRF images (left panel). Scale bar, 5 µm.

Article Snippet: To measure IL-2 release, we used the Mouse IL-2 DuoSet ELISA kit (R&D Systems, DY402-05) using the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Derivative Assay

Journal: bioRxiv

Article Title: IRAK4 autophosphorylation controls inflammatory signaling by activating IRAK oligomerization

doi: 10.1101/2023.12.21.572799

Figure Lengend Snippet: a) IL-2 release in EL4-MyD88-GFP/IRAK4-KO cells reconstituted with IRAK4 WT , IRAK4 K213/14A and IRAK4 DD . IL-2 release was measured by ELISA 24 h after IL-1β stimulation. Values shown are the fold change in IL-2 release. Average values calculated from three independent experiments. Bars represent mean ± SEM. One way Anova was used to compare the replicate means. b) TIRF images and kymograph of EL4-MyD88-GFP/IRAK4-KO cells reconstituted with IRAK4 WT -mScarlet stimulated on IL-1 functionalized SLBs. Kymographs derived from red line overlaid TIRF images (left panel). Scale bar, 5 µm. c) Time-series TIRF images and fluorescence-intensity time series showing the formation of a single MyD88-GFP:IRAK4 WT assembly in EL4-MyD88-GFP/IRAK4-KO cells reconstituted with IRAK4 WT -mScarlet. Scale bar, 1 µm.

Article Snippet: To measure IL-2 release, we used the Mouse IL-2 DuoSet ELISA kit (R&D Systems, DY402-05) using the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Fluorescence

Journal: Journal of the American Society of Nephrology

Article Title: Complement Activation Regulates the Capacity of Proximal Tubular Epithelial Cell to Stimulate Alloreactive T Cell Response

doi: 10.1097/01.asn.0000135974.06478.7b

Figure Lengend Snippet: Figure 6. Detection of IL-2 and IFN- by ELISA. IFN-–stimulated PTEC were treated with NS or HIS for 30 min and subsequently co-cultured with T cells. (A and B) Supernatants were collected from co-culture of 2 105 PTEC with 2 105 T cells at various time points and assayed for IL-2 and IFN- by sandwich ELISA. P values are for comparisons between NS and HIS treatment. *P 0.05; **P 0.005; ***P 0.0003.

Article Snippet: Sandwich ELISA was performed using ELISA Kit for mouse IFN- or IL-2 (R&D Systems, Oxford, UK) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay, Sandwich ELISA

Journal: Journal of the American Society of Nephrology

Article Title: Complement Activation Regulates the Capacity of Proximal Tubular Epithelial Cell to Stimulate Alloreactive T Cell Response

doi: 10.1097/01.asn.0000135974.06478.7b

Figure Lengend Snippet: Figure 7. Relative involvement of CD4 and CD8 T cells in complement-enhanced alloreactive T cell response. IFN-–stimulated and NS- or HIS-treated PTEC were co-cultured with purified allo- reactive CD4 or CD8 T cells. Supernatants were collected from co-culture of PTEC with T cells after 72 h of co-culturing and assayed for IFN- by sandwich ELISA. P values are for comparisons between NS and HIS treatment. **P 0.005.

Article Snippet: Sandwich ELISA was performed using ELISA Kit for mouse IFN- or IL-2 (R&D Systems, Oxford, UK) according to the manufacturer’s instructions.

Techniques: Cell Culture, Purification, Co-Culture Assay, Sandwich ELISA

Journal: Journal of the American Society of Nephrology

Article Title: Complement Activation Regulates the Capacity of Proximal Tubular Epithelial Cell to Stimulate Alloreactive T Cell Response

doi: 10.1097/01.asn.0000135974.06478.7b

Figure Lengend Snippet: Figure 8. Complement-mediated PTEC stimulation of alloreactive T cell response can be inhibited with CTLA4-Ig. IFN-–stimulated and NS- or HIS-treated PTEC were co-cultured with purified alloreactive CD4 T cells in medium alone or in the presence of CTLA4-Ig (5 g/ml) or control Ig (human IgG1, 5 g/ml). Supernatants were collected from co-culture of PTEC with T cells after 72 h of co- culturing and assayed for IFN- by sandwich ELISA. P values are for comparisons between CTLA4-Ig treatment and medium alone or control Ig treatment. **P 0.005.

Article Snippet: Sandwich ELISA was performed using ELISA Kit for mouse IFN- or IL-2 (R&D Systems, Oxford, UK) according to the manufacturer’s instructions.

Techniques: Cell Culture, Purification, Control, Co-Culture Assay, Sandwich ELISA