Journal: Cell reports

Article Title: Evaluation of Single-Cell Cytokine Secretion and Cell-Cell Interactions with a Hierarchical Loading Microwell Chip

doi: 10.1016/j.celrep.2020.107574

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To prepare functionalized beads to detect IFN-γ or IL-2, a biotinylated antibody against human IFN-γ (Thermo Fisher, M701B) or a biotinylated antibody against human IL-2 (R&D Systems, BAF202) was conjugated to streptavidin polystyrene beads (mean size: 18.4 μm, Spherotech, SVP-200–4) or streptavidin magnetic beads (mean size: 21.7 μm, Spherotech, SVM-200–4) by shaking beads in 50 μg/mL IFN-γ antibody or IL-2 antibody solution at 4°C overnight in PBS, respectively.

Techniques: Recombinant, Labeling, Software

Journal: Cancer immunology research

Article Title: Antitumor effects of CAR T cells redirected to the EDB splice variant of fibronectin

doi: 10.1158/2326-6066.CIR-20-0280

Figure Lengend Snippet: (A) Scheme of retroviral vectors encoding the EDB-CAR, a 2A sequence, and truncated CD19 (tCD19); SH: short hinge; TM: transmembrane domain. (B-C) EDB-CAR expression was determined on T cells 7 days post-transduction by flow cytometric analysis. (B) Representative histogram (Black-filled line: NT T cells; Red-filled line: EDB-CAR transduced T cells) and (C) summary plot (n=7; Student’s t-test; ****p<0.0001). (D) NT and EDB-CAR T cells were incubated for 24 hours with increasing concentrations of rhFN-EDB-coated wells. IFNγ production in supernatants was determined by ELISA (n=4; two-way ANOVA; ***p<0.001; ****p<0.0001). (E) EDB expression of LM7, A673, A549, and U87 tumor cells, and two primary fibroblast cell lines (Fib 1, Fib 2) determined by RT-qPCR; dotted line represents the ΔCt score above which samples are considered positive. (F) NT and EDB-CAR T cells were incubated for 48 hours with EDB-positive tumor cells. IFNγ and IL2 production in supernatants was determined by ELISA (two-way ANOVA; ****p<0.0001). (G) Cytolytic activity of NT or EDB-CAR T cells at a E:T ratio of 4:1 against EDB-positive tumor cells (MTS assay; n=3; two-way ANOVA; *p<0.05; **p<0.01; ***p<0.001). (H) Cytolytic activity of NT or EDB-CAR T cells against primary fibroblasts (data of Fib 1 and Fib 2 was combined) at the indicated E:T ratios, with A549 serving as controls (n=3; two-way ANOVA; ****p<0.0001). (I) NT, EDB-CAR, or mutEDB-CAR T cells were incubated for 48 hours with U87 or U87FN−/− cells. IFNγ production in supernatants was determined by ELISA (n=3; two-way ANOVA; ****p<0.0001). (J) Cytolytic activity of NT, EDB-CAR, or mutEDB-CAR T cells against U87 or U87FN−/− cells (n=3; two-way ANOVA; ****p<0.0001). Mean+SEM is shown in panels.

Article Snippet: Cytokines were measured using human IFNγ and IL2 ELISA kits (R&D Systems).

Techniques: Retroviral, Sequencing, Expressing, Transduction, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Activity Assay, MTS Assay

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: Molecular Medicine Reports

Article Title: Continuous expression of CD83 on activated human CD4 + T cells is correlated with their differentiation into induced regulatory T cells

doi: 10.3892/mmr.2015.3796

Figure Lengend Snippet: Time-dependent expression of CD83, proliferation and the production of IL-2 and IFN-γ in CD4 + T cells. Following purity identification, the purified CD4 + T cells were either (A) directly stained with PE-labeled CD83 or (B–D) stimulated with anti-CD3/CD28 for 1–3 days, followed by staining with PE-labeled CD83. All samples were analyzed by flow cytometry and the typical flow cytometric histograms indicating the percentages of CD83-positive cells are shown. (E) Additionally, cell proliferation at days 1, 2 and 3 were determined using Cell Counting Kit 8. (F) The levels of IL-2 and IFN-γ in the supernatants from 1-, 2- and 3-day cultures of activated CD4 + T cells were analyzed by ELISA. Values are expressed as the mean ± standard deviation of three independent experiments ( ** P<0.01; * P<0.05). PE, phycoerythrin; CD, cluster of differentiation; IFN, interferon; IL, interleukin; neg.con, negative control.

Article Snippet: The levels of IL-2 and IFN-γ in CD4 + T-cell culture supernatants were measured in duplicate for each of the serial aliquots using a Human IL-2 Quantikine ELISA kit (cat. no. D2050) and Human IFN-γ Quantikine ELISA kit (cat. no. DIF50), purchased from R&D Systems (Minneapolis, MN, USA), according to the manufacturer's instructions.

Techniques: Expressing, Purification, Staining, Labeling, Flow Cytometry, Cell Counting, Enzyme-linked Immunosorbent Assay, Standard Deviation, Negative Control

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