mouse elispot development module  (R&D Systems)

Journal: Vaccines

Article Title: Immunogenic Comparison of Nucleic Acid-Based Vaccines Administered by Pyro-Drive Jet Injector

doi: 10.3390/vaccines12070757

Figure Lengend Snippet: Comparison of PJI-delivered pDNA and mRNA vaccines on OVA expression and immune response. ( A ) LUC-mRNA (0.2 or 1 µg/20 µL) or CpG-free pDNA encoding LUC (10 and 50 µg/20 µL) were injected intradermally into C57BL/6NJcl mouse backs by PJI ( n = 5 in each group), and luciferase activity in the injected skin tissues was analyzed 3, 6, and 24 h after the injection. Luciferase activity is represented by RLU. ( B ) OVA protein expression 24 h after the intradermal injection of the OVA-encoding CpG-free pDNA (50 µg/20 µL) or mRNA (0.2 or 1 µg/20 µL) into the BALB/c mouse skin ( n = 3 in each group). ( C ) Time course of the experiment. The BALB/c mice were vaccinated twice (prime and boost) intradermally using PJI at a two-week interval and the anti-OVA antibody titer was analyzed at four weeks and IFN-γ ELISpot at five weeks. ( D ) Anti-OVA antibody titer at four weeks after the intradermal prime/boost injections of OVA pDNA (50 µg/20 µL) or mRNA (0.2 or 1 µg/20 µL) into the BALB/c mouse or non-vaccinated control mouse skin by PJI ( n = 5 in each group). ( E ) OVA-specific IFN-γ-secreting splenocytes at five weeks after intradermal prime/boost injections of OVA pDNA (50 µg/20 µL) or mRNA (1 µg/20 µL) into the BALB/c mouse skin by PJI or non-vaccinated mice ( n = 5 in each group). All the results are shown as the mean ± SEM. The p -values were analyzed using the one-way ANOVA test adjusted for multiple testing using Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Mouse IFN-γ ELISpot assay (R&D Systems, Inc., Minneapolis, MN, USA) was performed according to the manufacturer’s instructions.

Techniques: Comparison, Vaccines, Expressing, Injection, Luciferase, Activity Assay, Enzyme-linked Immunospot, Control

Journal: ImmunoTargets and Therapy

Article Title: Deciphering Natural Killer Cell Cytotoxicity Against Medulloblastoma in vitro and in vivo: Implications for Immunotherapy

doi: 10.2147/ITT.S458278

Figure Lengend Snippet: Conjugation, granule polarization and exocytosis as steps of the cytotoxicity mechanism against MB. ( A ) Microscopy imaging (40X) of NK cells conjugation with K562, or MB cell lines. NK cells, target cells and lysosomal granules were respectively stained in green(CBG), red (CTO) and blue (LV). White arrows indicate conjugation area of NK cells with target cells in the upper line, and lysosomal granules polarization in the bottom line. ( B ) Flow cytometry gating strategy showing conjugated cells (CD56+ PVR+) among NK (CD56+) and MB cells (PVR+) co-culture. The upper line shows the FSC/SSC gate excluding cell debris showing NK cells alone (left panel), MB cells alone (middle panel) and conjugates (right panel) and the lower line shows conjugated cells in the CD56+/PVR+ Q2 quadrant. ( C ) Percentages of conjugated NK cells among total (unstimulated) fresh or expanded NK cells with K562, DAOY, D283 and D341 (ANOVA test; * p<0.05). ( D ) Expanded NK cells granzyme B secretion after co-culture with MB target cell lines at various E:T ratios (100:1, 50:1, 25:1), ELISPOT results expressed in Spot Forming Colony (SFC) per 10 5 NK cells (n=3).

Article Snippet: NK cell granzyme B degranulation was assessed using the Granzyme B Elispot Development Module (R&D Systems, USA) according to the manufacturer’s instructions.

Techniques: Conjugation Assay, Microscopy, Imaging, Staining, Flow Cytometry, Co-Culture Assay, Enzyme-linked Immunospot

Journal: Lupus science & medicine

Article Title: Impaired regulatory function of granzyme B-producing B cells against T cell inflammatory responses in lupus mice.

doi: 10.1136/lupus-2023-000974

Figure Lengend Snippet: Figure 1 B cells from the mice spleen produced granzyme B (GrB) spontaneously. (A) Spleen single-cell suspensions were isolated from B6 mice and incubated with brefeldin A (BFA) (10 µg/mL), ionomycin (1 µg/mL) and phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) for 5 hours. The expression of GrB in CD19+ B cells was detected by staining with anti-CD19, anti- CD3ε, anti-CD49b and anti-GrB. FACS gating strategy for identifying the expression of GrB on CD19+ B cells was shown. (B) Flow cytometry-sorted CD19+ B cells (1×106) from the spleen of B6 mice were set to detect the mRNA expression of GrB by PCR. (C) Freshly purified CD19+ B cells (2 × 105; middle) from B6 spleen were cultured with CpG (10 µg/mL) stimulation on mice GrB-specific ELISpot plates for 24 hours. Medium (left) and CD8a+ T cells (right) were used as blank control and positive control, respectively. Dots were counted and the representative of independent data from five different B6 mice was shown (p<0.001). ***p<0.001 (Student’s t-test C).

Article Snippet: Mouse GrB Antibody (Cat# AF1865), Normal Goat IgG Control (Cat# AB- 108- C) and the Mouse GrB ELISpot Development Module (Cat# SEL1865) were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Produced, Isolation, Incubation, Expressing, Staining, Flow Cytometry, Purification, Cell Culture, Enzyme-linked Immunospot, Control, Positive Control

Journal: Lupus science & medicine

Article Title: Impaired regulatory function of granzyme B-producing B cells against T cell inflammatory responses in lupus mice.

doi: 10.1136/lupus-2023-000974

Figure Lengend Snippet: Figure 4 Reduced granzyme B (GrB)-producing Breg cells in lupus mice. Bm12 mice spleen lymphocytes (1.2×108 cells) were injected intravenously into indicated animals (aged 6–8 weeks). Representative anti-ANAs (p<0.001) (A) staining and ELISA analysis of anti-double-stranded DNA (anti-dsDNA) (p=0.002) (B) of serum from mice described in A–B at 14 days. (C) The frequencies of GrB-producing Breg cells were assayed by flow cytometry in lupus (n=10), and naïve mice (n=10), the representative dots (left) and statistical results were shown (right) (p=0.001). Purified CD19+ B cells from lupus (n=5) and naïve mice (n=5) were subjected to detection of mRNA expression of GrB by PCR (left) (D) and quantitative PCR (right) (p=0.037) (E). CD19+ B cells (2.5×105 cells/well) from lupus (n=5) and naïve mice (n=5) were cultured with CpG stimulation (10 µg/mL) on specific mice GrB ELISpot plates for 24 hours. The representative figures (left) and statistical results (right) were shown (p<0.001) (F). *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test C, E, F and Mann-Whitney U test A, B).

Article Snippet: Mouse GrB Antibody (Cat# AF1865), Normal Goat IgG Control (Cat# AB- 108- C) and the Mouse GrB ELISpot Development Module (Cat# SEL1865) were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Injection, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Purification, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Recombinant Pichinde viral vector expressing tuberculosis antigens elicits strong T cell responses and protection in mice

doi: 10.3389/fimmu.2023.1127515

Figure Lengend Snippet: Evaluation of TBvac2-induced T cell responses by ELISpot assay. (A) BL6 mice (N=3) were primed with 1x10 5 PFU of rP18tri viral vector (V) alone or TBvac-2 through the IM route, and 21 days later boosted with the same virus at the same dose through the IN route. At 7 d post-boost (dpb), splenocytes were isolated and incubated with medium alone or peptide pools of Ag85B or ESAT-6/EsxA, followed by detection of mouse IFNγ by ELISpot. (B) Representative ELISpot wells. (C) The number of IFNγ-secreting cells per million splenocytes in the vector and TBvac-2-immunized mice after Ag85B or ESAT-6/EsxA peptide stimulation was normalized with medium-alone control and shown as the average with standard deviation. Statistical analysis was conducted with unpaired t-test. **p < 0.01.

Article Snippet: ELISpot was performed using Mouse IFNγ ELISpot Development Module (R&D Systems, SEL485) following the manufacturer’s instruction.

Techniques: Enzyme-linked Immunospot, Plasmid Preparation, Virus, Isolation, Incubation, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Recombinant Pichinde viral vector expressing tuberculosis antigens elicits strong T cell responses and protection in mice

doi: 10.3389/fimmu.2023.1127515

Figure Lengend Snippet: rP18tri-based TB vaccines induced multifunctional CD4 T cells. (A) BL6 mice (N=5) were immunized with rP18tri vector (V), TBvac-2, and TBvac-10 through IN-IN prime-boost strategy. At 7 dpb, PBMCs were collected and incubated in medium only or with pooled Ag85B/ESAT6 antigens, and stained for cell surface markers and cytokines. Representative flow cytometry plots of CD4 T cells (CD44 h CD4 + CD3 + ) positive for dual cytokine expression (IFNγ/IL-2, IFNγ/TNFα, and IL-2/TNFα) after either medium or peptide stimulation were shown for each immunization group (B) . After normalization with the medium control, the average percentages of dual- and triple-positive (IFNγ/IL-2/TNFα) CD4 T cells after peptide stimulation were shown for each group (C) . *p<0.05.

Article Snippet: ELISpot was performed using Mouse IFNγ ELISpot Development Module (R&D Systems, SEL485) following the manufacturer’s instruction.

Techniques: Vaccines, Plasmid Preparation, Incubation, Staining, Flow Cytometry, Expressing, Control