his-tag protein elisa kit  (Advisains)

Journal: Virulence

Article Title: Staphylococcus aureus planktonic but not biofilm environment induces an IFN-β macrophage immune response via the STING/IRF3 pathway

doi: 10.1080/21505594.2023.2254599

Figure Lengend Snippet: Differences in bacterial gene expression and release of 3’3’-cGAMP or protein a between SA planktonic and biofilm cultures. SA was cultured in DMEM high glucose with 10% heat-inactivated FCS at 37°C and 5% CO 2 either under shaking for planktonic or static for biofilm conditions. Bacteria and supernatants were harvested after 24 hours of planktonic or after 1 day, 3 days or 6 days of biofilm culture. a) Gene expression analysis of c-di-AMP synthase (diadenylate cyclase, DacA), c-di-GMP synthase (GGDEF domain protein, diguanylate cyclase, DGC) and phosphodiesterase (GdpP). mRNA levels of dacA , dgc and gdpP were quantified by RT-qPCR. Data are presented as relative gene expression of gene of interest related to the reference gene gyrB . n = 3 experiments. b) 3’3’-cGAMP release by planktonic and biofilm SA. 3’3’-cGAMP concentration was measured in medium without bacteria, SA planktonic CM and 6 days biofilm CM. For validation of the assay, known concentrations of 3’3’-cGAMP (0, 100, 1000 and 10,000 ng/ml) in medium were included in the measurement. Concentrations were quantified in the supernatants by competitive ELISA. Data are presented as absolute concentration (ng/ml). n = 3 different CM batches in duplicates. c+d) Gene expression analysis of agr/SarA quorum sensing (QS) molecules. mRNA levels of the agr effector molecule RNAIII and RNAIII encoded PSM δ-toxin ( hld gene, c) and SarA regulator protein sarS (d) were quantified by RT-qPCR. Data are presented as relative gene expression of gene of interest related to the reference gene gyrB . n = 3 experiments. e) Protein A production by planktonic and biofilm SA. mRNA levels of spa were quantified by RT-qPCR (left graph). Data are presented as relative gene expression of gene of interest related to the reference gene gyrB . n = 3 experiments. Protein A concentration was measured in SA planktonic CM and 6 days biofilm CM (right graph). For validation of the assay, known concentrations of Protein A (SpA; 50 and 100 pg/ml) in medium were included in the measurement. Concentrations were quantified in the supernatants by quantitative sandwich ELISA. Data are presented as absolute concentration (ng/ml). n = 3 different CM batches in duplicates. For all: Data are presented as mean ± SEM and single values are shown as dots. p-values were calculated by ordinary one-way ANOVA with post-hoc Bonferroni corrected multiple comparison. # is showing significance between different CM. # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: Protein A was quantified in the CM using a Protein A ELISA Kit (Abcam, UK) according to the manufacturer’s protocol.

Techniques: Gene Expression, Cell Culture, Bacteria, Quantitative RT-PCR, Concentration Assay, Biomarker Discovery, Competitive ELISA, Sandwich ELISA, Comparison

Journal: Nature Communications

Article Title: Modular-designed engineered bacteria for precision tumor immunotherapy via spatiotemporal manipulation by magnetic field

doi: 10.1038/s41467-023-37225-1

Figure Lengend Snippet: A Serum levels of TNF-α, IL-6, IFN-γ, and IFN-α1, as analyzed by ELISA on day 9 in the indicated groups introduced in Fig. ( n = 5). Cytokine concentration of each sample was normalized to Z-score (i.e., standard score), which was calculated as ( X – E[ X ])/σ[ X ]. X , the cytokine concentration of the individual sample; E[ X ], the average concentration of all samples; σ[ X ], the standard deviation of cytokine concentrations of all samples. B Ifna mRNA expression levels in tumor-infiltrating DCs, as determined by RT-qPCR on day 9 ( n = 5 mice). C Scheme and grouping of in vivo therapy to evaluate the role of the type I IFN signaling pathway and CD8 + T cells. The colons of BALB/c mice were inoculated with CT-26-luc cells on day -7, and the mice were treated with engineered bacteria (1 × 10 8 CFU) by colon-specific administration on day 0 and day 3, followed by AMF treatment (310 kHz and 23.8 kA/m) for 80 min at 24 h after colon administration. Anti-CD8 (15 mg/kg, clone TIB210) or anti-IFNAR1 (15 mg/kg, clone R46A2) neutralization antibodies were i.p . injected on days −9, −6, −3, 0, and 3. D Bioluminescence imaging was performed to evaluate tumor growth on day 0, 7, and 14 ( n = 5). E Survival curves of mice from the indicated groups for 80 days ( n = 5 mice). F Semi-quantitative results of bioluminescence intensity in the tumor region on day 14, which were normalized to day 0 ( n = 5 mice). G Schematic illustration of the type I IFN pathway and adaptive immunity activated by AMF-Bac. TLRs, Toll-like receptors. The data ( B , E , F ) are shown as the mean ± SD. Statistical analysis was performed by a two-tailed unpaired t test. Survival significance was analyzed by the log-rank test. * P < 0.05; ** P < 0.01 ; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: The His-tag Protein ELISA kit (catalog No. ab285248) and mouse IFN-α1 ELISA kit (catalog No. ab252352) were purchased from Abcam (UK).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation, Expressing, Quantitative RT-PCR, In Vivo, Neutralization, Injection, Imaging, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: A genetically engineered, stem-cell-derived cellular vaccine

doi: 10.1016/j.xcrm.2022.100843

Figure Lengend Snippet: CRISPR genetic engineering of an iPSC line to create an immunogenic, self-adjuvanting cellular vaccine (A) Universal vaccine cell CRISPR genetic engineering strategy to create an apoptotic cellular vehicle for antigen delivery. (B and C) Representative flow cytometric analysis showing expression of SARS-CoV-2 WA1/2020 spike protein on the cell surface (B) and by western blot showing spike protein within UVC whole-cell lysates (C). (D) ELISA quantification of spike protein released upon UVC lysis for 2 independent UVC cultures. (E) Proportion of apoptotic cells at 24 and 72 h post-irradiation as measured by 7-AAD staining and flow cytometry. (F) Morphology, observed by light microscopy, of engineered UVC during expansion culture and, when reseeded into culture 24 h after irradiation, showing apoptosis and cell death. (G) Absence of detectable proliferation of irradiated UVC as determined by CellTrace yellow proliferation dye staining and measuring the dilution of the dye by flow cytometry over 72 h. (H) Representative flow cytometric analysis showing deletion of MHC class I and overexpression of MICA on the UVC surface by CRISPR engineering. (I) Cell counts of 3 independent cultures showing exponential expansion of live engineered UVCs over 21 days in culture. (J) Relative expression of pluripotency and self-renewal genes by UVC and the control iPSCs from which they were derived, as measured by quantitative PCR, showing maintenance of an iPSC gene expression profile after genetic engineering and expansion. Error bars represent mean ± SEM of 3 technical replicas. All experiments were repeated at least three times.

Article Snippet: S1 Spike protein was detected using a Covid-19 S-protein ELISA kit (Abcam) specific to S1RBD.

Techniques: CRISPR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Lysis, Irradiation, Staining, Flow Cytometry, Light Microscopy, Over Expression, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: A genetically engineered, stem-cell-derived cellular vaccine

doi: 10.1016/j.xcrm.2022.100843

Figure Lengend Snippet:

Article Snippet: S1 Spike protein was detected using a Covid-19 S-protein ELISA kit (Abcam) specific to S1RBD.

Techniques: Variant Assay, Formalin-fixed Paraffin-Embedded, Recombinant, Activation Assay, Protease Inhibitor, Purification, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Luciferase, BIA-KA, Expressing, Plasmid Preparation, Software