mouse ifn γ elispot plus plates  (Mabtech Inc)

Journal: Infection and Immunity

Article Title: Construction and characterization of novel Mycobacterium tuberculosis -derived triple and quadruple knockout vaccines against tuberculosis

doi: 10.1128/iai.00500-25

Figure Lengend Snippet: Rational deletion of genes reduces the intracellular survival and increases the processing of live vaccines through PL fusion and autophagy. ( A ) Deletion of genes in Mtb reduces intracellular viability/growth. BMDMs from naïve C57BL/6 mice were activated with IFN-γ and infected (MOI 1:1) with Mtb strains H37Rv, DKO, TKO-Z, TKO-D, or QKO and incubated. After 1, 4, and 8 days post-infection, cells were washed, lysed, and plated for viable colony counts (CFUs). Data represent the mean ± SD CFUs from triplicate. ( B ) Deletion of genes in Mtb leads to efficient processing through PL fusion. BMDMs from naïve C57BL/6 mice were infected with (a–e) rfpH37Rv, rfpDKO, rfpTKO-D, rfpTKO-Z, and rfpQKO (MOI = 1:1) for 4 h, washed, incubated for 24 h, and stained with primary antibodies to phagosomal maturation marker Rab7, followed by staining with FITC (green) conjugated secondary antibody. Red fluorescent Mtb colocalizing with Rab7 antibodies was scored using a Nikon TiE Fluorescence Microscope and Metaview Deconvolution Software. ( C ) Graph showing percent colocalization of Mtb with Rab7. Percent colocalization was determined by counting 50 macrophages per well, each with 1–3 mycobacteria, and averaging counts from triplicate chambers. One of three similar experiments is shown. ( D ) Deletion of genes in Mtb leads to efficient processing through autophagy. BMDMs from naïve C57BL/6 mice were infected with (a–e) rfpH37Rv, rfpDKO, rfpTKO-D, rfpTKO-Z, and rfpQKO (MOI = 1:1) for 4 h, washed, incubated for 24 h, and stained with primary antibodies to autophagy marker LC3, followed by staining with FITC (green) conjugated secondary antibody. Red fluorescent Mtb colocalizing with LC3 antibodies was scored using a Nikon TiE Fluorescence Microscope and Metaview Deconvolution Software. ( E ) The graph shows the percent colocalization of Mtb with LC3. Percent colocalization was determined by counting 50 macrophages per well, each with 1–3 mycobacteria, and averaging counts from triplicate chambers. One of three similar experiments is shown. Statistical significance was calculated using Student’s t-test. P values below 0.05 ( P < 0.05) are considered significant.

Article Snippet: Mouse IFN-γ ELISpot Plus plates (Catalog #3321-4APT-10, MABTECH Inc., Cincinnati, OH) were washed three times with sterile PBS and then blocked with culture media (RMPI with 10% FBS).

Techniques: Vaccines, Infection, Incubation, Staining, Marker, Fluorescence, Microscopy, Software

Journal: Infection and Immunity

Article Title: Construction and characterization of novel Mycobacterium tuberculosis -derived triple and quadruple knockout vaccines against tuberculosis

doi: 10.1128/iai.00500-25

Figure Lengend Snippet: Rational deletion of genes in Mtb increases the immunogenicity of vaccine strains. ( A ) In vitro antigen presentation. BMDMs from naïve C57BL/6 mice were infected with BCG and Mtb strains (H37Rv, DKO, TKO-D, TKO-Z, and QKO) (MOI = 1:5) for 4 h and cocultured with BB7 T-cell hybridoma specific for Ag85B 241-256 peptide. After 16 h, culture fluids were collected and assayed for IL-2 levels released by the BB7 cells in response to Ag85B peptide. ( B ) Ex vivo immunogenicity to vaccine strains. C57BL/6 mice were vaccinated with BCG, DKO, TKO-D, TKO-Z, and QKO vaccine strains (1 × 10 6 subcutaneously) and the control H37Rv. After 30 days of post-immunization, mice were euthanized, and spleens were isolated. Splenocytes (2.5 × 10 5 /well) were plated and stimulated in vitro for 48 h with Mtb H37Rv whole-cell lysate (20 µg/mL). Supernatants from cultures were collected, and IFN-γ, (a) IL-1β, (b) IL-2, (c) IL-12 (d), and TNF (e) levels were determined by ELISA. ELISpot analysis for IFN-γ-producing splenocytes in vaccinated mice (f). C57BL/6 mice were vaccinated with BCG, DKO, TKO-D, TKO-Z, and QKO vaccine strains and control H37Rv (1 × 10 6 subcutaneously). After 30 days post-immunization, mice were euthanized, and spleens were isolated. Splenocytes (2.5 × 10 5 /well) were plated and stimulated with a combination of Ag85B and CFP-10 peptides in vitro for 48 h. Ag85B/CFP-10 responsive IFN-γ-producing spleen cells were spotted using IFN-γ ELISpot plates following the manufacturer’s protocols. Statistical significance was calculated using Student’s t-test. P values below 0.05 ( P < 0.05) were considered significant.

Article Snippet: Mouse IFN-γ ELISpot Plus plates (Catalog #3321-4APT-10, MABTECH Inc., Cincinnati, OH) were washed three times with sterile PBS and then blocked with culture media (RMPI with 10% FBS).

Techniques: Immunopeptidomics, In Vitro, Infection, Ex Vivo, Control, Isolation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Science Advances

Article Title: Lipopolyplex-formulated mRNA cancer vaccine elicits strong neoantigen-specific T cell responses and antitumor activity

doi: 10.1126/sciadv.adn9961

Figure Lengend Snippet: ( A ). Treatment schedule for LPP-CT26 vaccination ( n = 5 mice per group) and correlative immune analysis. LN, lymph nodes. ( B to D ). Analysis of activation of DC subsets in lymph nodes after LPP-CT26 treatment. ( E ). Proportion of total CD4 + and CD8 + T cells in lymph nodes after LPP-CT26 immunization. ( F and G ). Proportion of CD4 + and CD8 + T cell activation in lymph nodes after LPP-CT26 immunization. ( H ). ELISpot assay of neoantigen-induced T cell (splenocyte) activation at the indicated time points. ( I ). Relative proportion of effector and memory T cells in lymph nodes at different time points upon LPP-CT26 vaccination. Data represent the means ± SD. One-way analysis of variance (ANOVA) test was performed for all comparisons. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). ns, not significant.

Article Snippet: Mouse IFN-γ ELISpot PLUS Kit (ALP) plates (Mabtech, 3321-4APT-10) were washed thoroughly five times with PBS before use.

Techniques: Activation Assay, Enzyme-linked Immunospot

Journal: Science Advances

Article Title: Lipopolyplex-formulated mRNA cancer vaccine elicits strong neoantigen-specific T cell responses and antitumor activity

doi: 10.1126/sciadv.adn9961

Figure Lengend Snippet: ( A to C ). Effects of LPP-mRNA vaccination routes on immune response and experimental lung metastasis. Shown are experimental schema for delivering the LPP-CT26 vaccine via three different administration routes ( n = 10 mice per route) (A); bar graphs presenting the end point (day 18) lung metastases in mice that received intravenously injected CT26-luc cells and LPP-CT26 vaccine administered via three different routes (B); and IFN-γ ELISpot assay of splenocytes from mice inoculated with the LPP-CT26 vaccine delivered via three routes (C). s.c., subcutaneous; i.m., intramuscular; i.d., intradermal; i.v., intravenous. ( D to F ). Effects of single- versus multisite LPP-mRNA vaccination on immune response and tumor burden. Shown are experimental schema with single or multiple vaccination sites ( n = 10 mice per group) (D); bar graphs presenting relative tumor burden assessed by fluorescent intensity of CT26-luc tumors in mice on day 10 (E); and IFN-γ ELISpot assay in splenocytes from mice with single or multisite LPP-CT26 vaccinations (F). ( G to I ). Effects of LPP-mRNA vaccine dosage on immune response and lung metastasis. Shown are experimental schema ( n = 10 mice per dose) (G); bar graphs presenting lung metastases in different LPP-CT26 dosage groups (H); and IFN-γ ELISpot assay of splenocytes in different LPP-CT26 dosage groups (I). Data were presented as means ± SD. One-way ANOVA statistical test was performed was performed for all data analysis. (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse IFN-γ ELISpot PLUS Kit (ALP) plates (Mabtech, 3321-4APT-10) were washed thoroughly five times with PBS before use.

Techniques: Injection, Enzyme-linked Immunospot

Journal: Science Advances

Article Title: Lipopolyplex-formulated mRNA cancer vaccine elicits strong neoantigen-specific T cell responses and antitumor activity

doi: 10.1126/sciadv.adn9961

Figure Lengend Snippet: ( A ) Experimental schema to assess the antitumor activity of LPP-MC38 in the MC38 tumor model. C57BL/6 mice ( n = 10 per group) were subcutaneously inoculated with MC38-luc tumor cells and then immunized with different doses of LPP-MC38 vaccines. ( B ) Survival rate in mice ( n = 10 mice per group) receiving eight immunizations with different doses of LPP-MC38 or irrelevant (GFP) LPP. ( C to E ) Effects of LPP-MC38 on primary tumor growth and the immune response. MC38-luc tumor cells were subcutaneously implanted in the left flanks of mice, and 3 days later, the mice were immunized with LPP-MC38 ( n = 5) or PBS ( n = 5) for a total of five injections (C). The end point (day 17) tumor images and weights were presented (D). IFN-γ ELISpot assay in splenocytes restimulated with the individual peptides (E). ( F to I ) Effects of LPP-CT26 on primary tumor growth and the immune response. Experimental schema to assess the antitumor activity of LPP-CT26 in BALB/c ( n = 10 per group) mice bearing subcutaneous CT26-luc tumors immunized with different doses of LPP-CT26 or irrelevant (GFP) LPP or PBS (F). Shown in (G) are the end point (day 16) CT26-luc tumor images and weights (means ± SD). Shown in (H) and (I) are IFN-γ ELISpot assays of splenocytes in CT26-bearing mice (H) and for a representative neoantigen in CT26 tumor tissue [ n = 5 (I)]. ( J ) Flow cytometric analysis of CD4 + and CD8 + T cells in bulk splenocytes. ( K ) ICS by fluorescence-activated cell sorting (FACS) in T cells in mouse bulk splenocytes treated with LPP-CT26 or PBS. ( L ) IFN-γ ELISpot assays for immunogenic neoantigens and their corresponding WT sequences. Data were presented as means ± SD. One-way ANOVA statistical test was performed for all data analysis. (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse IFN-γ ELISpot PLUS Kit (ALP) plates (Mabtech, 3321-4APT-10) were washed thoroughly five times with PBS before use.

Techniques: Activity Assay, Vaccines, Enzyme-linked Immunospot, Fluorescence, FACS, Immunopeptidomics

Journal: Science Advances

Article Title: Lipopolyplex-formulated mRNA cancer vaccine elicits strong neoantigen-specific T cell responses and antitumor activity

doi: 10.1126/sciadv.adn9961

Figure Lengend Snippet: ( A ) Experimental scheme and timeline for CD4 + /CD8 + T cell depletion experiments. BALB/c mice ( n = 6 per group) were administrated with T cell–depleting antibodies via intraperitoneal injection. CT26-luc cells were injected through tail vein on day 0 to establish the lung metastasis model. LPP-CT26 immunization was started from day 3. ( B ) In vivo imaging system (IVIS) images of CT26-luc tumors on day 14. ( C ) Fluorescence intensity of CT26-luc tumors in different groups on day 14. ( D ) Pictures of the lungs of mice at the time of euthanasia (day 18). ( E ) Quantification of the end point lung tumor nodules (day 18). ( F ) IFN-γ ELISpot assay of splenocytes from CT26-luc–bearing mice subjected to CD4 + or CD8 + T cell depletion. ( G ) FACS plots illustrating the effects of antibody-mediated depletion of CD4 + or CD8 + T cells in the mouse spleen. ( H and I ) ICS of IFN-γ by FACS in CD4 + /CD8 + T cells in bulk splenocytes after T cell depletion. ( J ) FACS gating strategy for assessing tumor cell death after coculture of tumor cells with T cells. ( K ) Representative FACS images of CT26-luc cell death upon coculture with T cells in the presence of the indicated neutralizing antibodies. ( L ) Percentage of CT26-luc cells killed after coculture with T cells in vitro via FACS assay. Data were presented as means ± SD. One-way ANOVA statistical test was performed for all data analysis (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse IFN-γ ELISpot PLUS Kit (ALP) plates (Mabtech, 3321-4APT-10) were washed thoroughly five times with PBS before use.

Techniques: Injection, In Vivo Imaging, Fluorescence, Enzyme-linked Immunospot, In Vitro

Journal: Science Advances

Article Title: Lipopolyplex-formulated mRNA cancer vaccine elicits strong neoantigen-specific T cell responses and antitumor activity

doi: 10.1126/sciadv.adn9961

Figure Lengend Snippet: ( A to G ) Experiments in the LPP-CT26 model. Shown in (A) is the experimental scheme for the two different LPP-CT26 immunization schedules, i.e., prophylaxis (Pro) before intravenous injection of the CT26-luc cells and vaccination 3 days after CT26-luc cell injection (post). BALB/c mice ( n = 9 or 10 per group) were immunized with 10 μg of LPP-CT26 in both settings. (B) to (E) show IVIS images (B) and fluorescence intensity of CT26-luc tumor burden in the lung (C) on day 10, the end point (day 12) lung images (D), and normalized IFN-γ ELISpot assay of splenocytes in CT26-luc lung metastasis mice (E). (F) Counts of neoantigen with and without immune response after LPP-CT26 prophylactic immunization versus post-immunization. (G). Percentage of CD4 + /CD8 + T cell subsets including Tem and Trm cells in the spleen from mice treated with prophylactical versus post-administration. ( H to L ) Experiments in the B16F10 model. Shown in (H) is the treatment schedule for post versus prophylactic administration of the LPP-B16 vaccines in C57BL/6 mice bearing the B16F10 lung metastases ( n = 10 per group). (I) Representative images of the end point lungs (left) and bar graphs of lung metastases (right) on day 18. (J) Normalized ex vivo IFN-γ ELISpot counts for the LPP-B16 neoantigens in splenocytes from indicated groups of mice ( n = 5 per group). (K) Counts of neoantigens with or without immune response after LPP-B16 prophylactic versus post-immunization. (L) Summary of immunogenic versus nonimmunogenic neoantigens in the three animal models. Data were presented as means ± SD. One-way ANOVA test was performed for all data analysis (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse IFN-γ ELISpot PLUS Kit (ALP) plates (Mabtech, 3321-4APT-10) were washed thoroughly five times with PBS before use.

Techniques: Injection, Fluorescence, Enzyme-linked Immunospot, Vaccines, Ex Vivo, Immunopeptidomics

Journal: Science Advances

Article Title: Lipopolyplex-formulated mRNA cancer vaccine elicits strong neoantigen-specific T cell responses and antitumor activity

doi: 10.1126/sciadv.adn9961

Figure Lengend Snippet: ( A ) Experimental scheme. Both treatment-naïve and MC38 tumor–regressing mice were inoculated with MC38 cells subcutaneously. Tumor growth was monitored, and mice were euthanized on day 24. ( B and C ) Tumor growth curves until day 27 (B) and the end point tumor weight (C). ( D ) Individual peptide restimulation for IFN-γ ELISpot assay in bulk splenocytes. ( E and F ) Representative Tcm, Tem, and Trm in the splenocytes as assayed by flow cytometry (E) and quantitative summary (F) of Tcm, Tem, and Trm in T cell subtypes in the splenocytes from the treatment-naïve and rechallenged mice. Data were presented as means ± SD. One-way ANOVA statistical test was performed for all data analysis (* P < 0.05, ** P < 0.01).

Article Snippet: Mouse IFN-γ ELISpot PLUS Kit (ALP) plates (Mabtech, 3321-4APT-10) were washed thoroughly five times with PBS before use.

Techniques: Enzyme-linked Immunospot, Flow Cytometry

Journal: Science Advances

Article Title: Lipopolyplex-formulated mRNA cancer vaccine elicits strong neoantigen-specific T cell responses and antitumor activity

doi: 10.1126/sciadv.adn9961

Figure Lengend Snippet: ( A ) Diagram of the clinical design for the LPP-PCV treatment. ( B and C ) IFN-γ ELISpot assay of PBMCs after different cycles of LPP-PCV immunization from patient 05002 (B) and patient 04029 (C). ( D ) Representative MRI images of two major target lesions (TL1 was highlighted in red, and TL2 was highlighted in yellow) in patient 05002. Note that 5 months after vaccination, the diameters of both TLs were reduced. ( E and F ) FACS plots (E) and bar graphs (F) showing the LPP-PCV–induced increases in patient 04029 PBMCs of both CD4 + and CD8 + T cells positive for de novo–primed neoantigen #1 tetramers. Data were presented as means ± SD. One-way ANOVA statistical test was performed for all data analysis (* P < 0.05).

Article Snippet: Mouse IFN-γ ELISpot PLUS Kit (ALP) plates (Mabtech, 3321-4APT-10) were washed thoroughly five times with PBS before use.

Techniques: Enzyme-linked Immunospot

Journal: NPJ Vaccines

Article Title: T-cell immunity induced and reshaped by an anti-HPV immuno-oncotherapeutic lentiviral vector

doi: 10.1038/s41541-024-00894-0

Figure Lengend Snippet: a Timeline of single injection or prime-boost immunization with 1 × 10 9 TU of Lenti-HPV-07 ind or Lenti-HPV-07 nj , antibody and T-cell assays. b , c ED50 comparison of anti-VSV-G ind neutralizing antibodies in sera from mice primed with Lenti-HPV-07 ind and boosted homologously with Lenti-HPV-07 ind or heterologously with Lenti-HPV-07 nj . ED50 against VSV-G ind ( b ) or against VSV-G nj ( c ) ( n = 4/group). Statistical significance was determined using a repeated measures (RM) two-way ANOVA (* p < 0.05, *** p < 0.001, **** p < 0.001). IFN-γ ELISPOT responses of T splenocytes from the same individual mice were studied at the indicated time points after in vitro stimulation with ( d ) six peptide pools spanning the full sequence of VSV-G ind , or ( e ) four peptide pools spanning the sequence of detoxified E6 HPV16 , E7 HPV16 , E6 HPV18 or E7 HPV18 , as encoded by Lenti-HPV-07. f IFN-γresponses of T splenocytes of mice assessed at D14 after a mono-injection with 1 × 10 9 TU of Ctrl lenti ind, Lenti-HPV-07 ind or Lenti-HPV-07 ind heat-inactivated at 70 °C during 1 h ( n = 6). Naive mice served as control ( n = 2). Stimulation was performed using either the indicated lentiviral vectors or peptides both at 4 µg/ml. SFU spot forming unit. Statistical significance was determined by Mann–Whitney t -test (ns not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: Splenocytes were then plated at 1 × 10 5 cells/well in 200 μl of RPMI-GlutaMAX, containing 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin, 1 × 10 –4 M non-essential amino-acids, 1% vol/vol HEPES, 1 × 10 –3 M sodium pyruvate and 5 × 10 –5 M of β-mercaptoethanol in ELISPOT plates (Mouse IFN-γ ELISPOT PLUS , Mabtech).

Techniques: Injection, Comparison, Enzyme-linked Immunospot, In Vitro, Sequencing, Control, MANN-WHITNEY

Journal: Veterinary Sciences

Article Title: Development of Virus-like Particle Plant-Based Vaccines against Avian H5 and H9 Influenza A Viruses

doi: 10.3390/vetsci11020093

Figure Lengend Snippet: Interferon-γ ELISpot results for spleen cells restimulated with either H5 or H9 VLPs or negative control plant lysate (NC) at 0.9 µg/mL. Cell-only control wells had a maximum of 6 spots. The results were calculated as mean (±SEM) of duplicate wells.

Article Snippet: The precoated ELISpot Plus: Mouse IFN-γ (ALP) 96-well plates (Mabtech, Stockholm, Sweden) were incubated at room temperature for 30 min with 10% RPMI media without 2-mercaptoethanol before the media were discarded.

Techniques: Enzyme-linked Immunospot, Negative Control, Control