murine ifnγ single color enzymatic elispot assay  (Cellular Technology Ltd)

Journal: bioRxiv

Article Title: An Effective mRNA-LNP Vaccine Against the Lethal Plague Bacterium

doi: 10.1101/2022.08.07.503096

Figure Lengend Snippet: ( A ) Western blot analysis of F1 expression in HeLa cells transfected with SP- caf1 mRNA-LNPs (2 μg/mL, 24−96 h). Purified recombinant F1 (20ng) was used as reference for protein migration ( B ) Schematic diagram of immunization and sample collection. C57BL/6 mice were immunized with 5µg SP- caf1 or Luciferase mRNA-LNPs or with 80µg recombinant F1 protein with alum and boosted with an equivalent dose 21 and 42 days later. Serum samples were collected at days 20, 41 and 56. Spleens were collected at day 63. Illustration created with BioRender.com. ( C ) F1-specific cellular response determined by ELISpot. Splenocytes from vaccinated animals were stimulated with 10µg/ml F1 peptide mix for 24hrs, and the frequency of IFNγ-secreting cells was determined using the ELISpot assay. ( D ) Anti-F1 IgG titers were detected from serum samples at the indicated time points via an ELISA assay (n=3). Statistical analysis was performed using a one-way ANOVA followed by post hoc Newman–Keuls test (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: Cells were incubated with antigens for 24 h, and the frequency of IFNγ-secreting cells was determined using Murine IFNγ Single-Color Enzymatic ELISPOT kit (CTL, No. MIFNG 1M/5) with strict adherence to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Transfection, Purification, Recombinant, Migration, Luciferase, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: An Effective mRNA-LNP Vaccine Against the Lethal Plague Bacterium

doi: 10.1101/2022.08.07.503096

Figure Lengend Snippet: ( A ) Western blot analysis of HeLa cells transfected with SP- caf1 -hFc and ΔSP- caf1 mRNA-LNPs (0.5 μg/mL, 72 h). ( B ) Schematic diagram of immunization and sample collection. C57BL/6 mice were immunized with mRNA-LNPs encapsulating SP- caf1 -hFc, ΔSP- caf1 or luciferase (5µg) or with 80µg recombinant F1 protein with alum and boosted with an equivalent dose 14 and 28 days later. Serum samples were collected at days 13, 27 and 42. Spleens were collected at day 42. Illustration created with BioRender.com. ( C ) F1-specific cellular response determined by ELISpot. Splenocytes from vaccinated animals were stimulated with 10µg/ml F1 peptide mix for 24hrs, and the frequency of IFNγ-secreting cells was determined using the ELISpot assay (n=4). ( D ) Anti-F1 IgG titers were detected from serum samples at the indicated time points via an ELISA assay (n=12). Statistical analysis was performed using a one-way ANOVA followed by post hoc Newman–Keuls test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: Cells were incubated with antigens for 24 h, and the frequency of IFNγ-secreting cells was determined using Murine IFNγ Single-Color Enzymatic ELISPOT kit (CTL, No. MIFNG 1M/5) with strict adherence to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Luciferase, Recombinant, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

Journal: International Immunology

Article Title: Immunological association of inducible bronchus-associated lymphoid tissue organogenesis in Ag85B-rHPIV2 vaccine-induced anti-tuberculosis mucosal immune responses in mice

doi: 10.1093/intimm/dxy046

Figure Lengend Snippet: Ag85B is not essential for the induction of iBALT organogenesis by HPIV2. (A–D) Mice were intra-nasally administered HPIV2, two times at a 1-week interval. Seventeen days after the final immunization, lung samples were examined by means of immunohistochemistry (A, B) and flow cytometry (C, D). (A, B) Lung tissues were prepared and stained with the indicated antibodies and reagent. Data are representative of two independent experiments. Scale bars: 300 µm (A) and 100 µm (B). Dotted circles in (A) indicate individual iBALT structures. Arrows in (B) indicate CD11b + CD11c + cells. (C) Representative flow cytometric data of the lung inoculated with either HPIV2 or PBS as a control for the evaluation of cytokine production. Zombie − TCRβ + CD4 + populations are shown. (D) Numbers of T h 17 (gated on Zombie − TCRβ + CD4 + IL-17A + ) and T h 1 (gated on Zombie − TCRβ + CD4 + IFNγ + ) cells in the lung were determined on the basis of total cell numbers and flow cytometric data. Data are combined from two independent experiments, and each point represents data from an individual mouse. Horizontal lines indicate medians. Statistical significance was evaluated by using the Mann–Whitney test; ** P < 0.01; N.S., not significant.

Article Snippet: IFNγ ELIspot assay was performed by using a Murine IFNγ Single-color Enzymatic ELIspot Assay Kit (Cellular Technology Limited, Cleveland, OH, USA) in accordance with the manufacturer’s instructions.

Techniques: Immunohistochemistry, Flow Cytometry, Staining, Control, MANN-WHITNEY

Journal: International Immunology

Article Title: Immunological association of inducible bronchus-associated lymphoid tissue organogenesis in Ag85B-rHPIV2 vaccine-induced anti-tuberculosis mucosal immune responses in mice

doi: 10.1093/intimm/dxy046

Figure Lengend Snippet: Lymphotoxin signals are essential for iBALT organogenesis and Ag85B-specific immune responses in the lung. Mice were intra-nasally administered Ag85B-rHPIV2 four times at 2-week intervals with or without LTβR-Fc administration. Lung samples were analyzed 2 weeks after the final immunization with Ag85B-rHPIV2. (A) Lung cells were prepared and examined by flow cytometry. The percentage of GL7 + cells in the B220 + B-cell population is shown. Data are combined from four independent experiments, and each point represents data from an individual mouse. (B) Lung tissues were prepared and examined by means of immunohistochemistry. Eight separate slides were prepared from each lung sample for the counting of iBALT structures. Data are combined from four independent experiments, and each point represents data from an individual mouse. In both plots, center values indicate the median. Statistical significance was evaluated by using one-way ANOVA; **** P < 0.0001; N.S., not significant. (C) Lung cells were prepared and then examined by means of IFNγ ELIspot assay. Pearson’s correlation analysis was conducted between the numbers of IFNγ spots and iBALT structures. (D) BALF and serum were prepared and examined by means of an ELISA for the production of Ag85B-specific IgA and IgG antibodies. Data are combined from four independent experiments and expressed as mean ± SEM ( n = 10–16 per group). Statistical significance was evaluated by using Student’s t -test; * P < 0.05; N.S., not significant.

Article Snippet: IFNγ ELIspot assay was performed by using a Murine IFNγ Single-color Enzymatic ELIspot Assay Kit (Cellular Technology Limited, Cleveland, OH, USA) in accordance with the manufacturer’s instructions.

Techniques: Flow Cytometry, Immunohistochemistry, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

Journal: International Immunology

Article Title: Immunological association of inducible bronchus-associated lymphoid tissue organogenesis in Ag85B-rHPIV2 vaccine-induced anti-tuberculosis mucosal immune responses in mice

doi: 10.1093/intimm/dxy046

Figure Lengend Snippet: CD11b + cells are essential for iBALT organogenesis and Ag85B-specific immune response in the lung. CD11b-DTR bone marrow chimera mice were intra-nasally administered Ag85B-rHPIV2 four times at 2-week intervals with or without diphtheria toxin injection. Lung samples were analyzed 2 weeks after the final immunization with Ag85B-rHPIV2. (A) Lung tissues were prepared and examined by means of immunohistochemistry. Eight separate slides were prepared from each lung sample for the counting of iBALT structures. (B) Lung cells were prepared and then analyzed by means of an IFNγ-ELIspot assay. In both plots, data are combined from four independent experiments and each point represents data from an individual mouse. Center values indicate the median. Statistical significance was evaluated by using one-way ANOVA; *** P < 0.001; * P < 0.05; N.S., not significant.

Article Snippet: IFNγ ELIspot assay was performed by using a Murine IFNγ Single-color Enzymatic ELIspot Assay Kit (Cellular Technology Limited, Cleveland, OH, USA) in accordance with the manufacturer’s instructions.

Techniques: Injection, Immunohistochemistry, Enzyme-linked Immunospot

Journal: International Immunology

Article Title: Immunological association of inducible bronchus-associated lymphoid tissue organogenesis in Ag85B-rHPIV2 vaccine-induced anti-tuberculosis mucosal immune responses in mice

doi: 10.1093/intimm/dxy046

Figure Lengend Snippet: Ag85B-specific immune response depends on the presence of iBALT. (A) Immunization schedule of Ag85B-rHPIV2. (i) Mice were nasally immunized with Ag85B-rHPIV2, two times only at early phase on day 0 and 7 and examined on day 24. (ii) Mice were nasally immunized with Ag85B-rHPIV2, four times as conventional method on day 0, 14, 28 and 42, and examined on day 56. (B) On day 9, lung tissues were prepared, stained with the indicated antibodies and reagent and then examined by means of fluorescence microscopy. Data are representative of two independent experiments. Scale bars: 100 µm. (C) Lung cells were prepared and then analyzed by means of an IFNγ-ELIspot assay. Data are representative pictures of two independent experiments ( n = 4–8 per group). (D) BALF was prepared and examined by means of an ELISA for the production of Ag85B-specific IgA antibody. Data are combined from two independent experiments and expressed as mean ± SEM ( n = 4 per group).

Article Snippet: IFNγ ELIspot assay was performed by using a Murine IFNγ Single-color Enzymatic ELIspot Assay Kit (Cellular Technology Limited, Cleveland, OH, USA) in accordance with the manufacturer’s instructions.

Techniques: Staining, Fluorescence, Microscopy, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay