mouse ifn γ elispot development module  (R&D Systems)

Journal: bioRxiv

Article Title: ULK1 drives NDP52-mediated selective autophagic degradation of MHC-I to promote immune evasion in HPV-positive head and neck cancer

doi: 10.64898/2026.03.14.711071

Figure Lengend Snippet: Immune cell profiling of tumor tissues from C57BL/6 mice injected with mEERL/sg-Scr or mEERL/sg2- Ulk1 (n=4/group). Isolated single cell suspensions were stained with an antibody cocktail and analyzed by flow cytometry. Percentages of total T cells of CD45 + (A), CD8 + T (B), and CD4 + T cells (C) of total CD3 + cells are shown. T cell profiling of tumor draining lymph nodes (TDLNs) from mice injected with either mEERL/sg-Scr or mEERL/sg- Ulk1 was isolated 21 days post-injection (n=3/group). Single-cell suspensions were stained with an antibody cocktail containing Zombie NIR, anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD44, and anti-CD62L antibodies. Representative bar graphs showing percentages of total T cells (D), CD4 + T (E), and CD8 + T cells (F), effector memory CD8 + T cells (G), and naïve CD8 + T cells (H). P -value was determined by One-way ANOVA. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. Cytolytic activity of isolated CD8 + T cells co-cultured with mEERL cells at a 50:1, 10:1, or 2:1 T cell: tumor cell ratios were measured by LDH assay (I). Effector functions of isolated CD8 + T cells were evaluated via IFNγ production measured by ELISA (J) and ELISpot (K and L). The number of spot-forming cells was counted using NIH ImageJ. All data shown are mean ± SEM and representative of at least 3 biological replicates. P -value was determined using Student’s t- Test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: IFN-γ production was determined using the Mouse IFN-γ ELISpot Development Module (R&D Systems, No. SEL485), visualized using the ELISpot Blue Color Module (Strep-AP and BCIP-BNBT) (R&D Systems, No. SEL002), and imaged using the ImmunoSpot Analyzer (CTL).

Techniques: Injection, Isolation, Single Cell, Staining, Flow Cytometry, Activity Assay, Cell Culture, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Notch1 blockade by a novel, selective anti-Notch1 neutralizing antibody improves immunotherapy efficacy in melanoma by promoting an inflamed TME.

doi: 10.1186/s13046-024-03214-5

Figure Lengend Snippet: Fig. 5 Anti-N1 promotes CD8+ T cytotoxicity. A, B) Flow cytometry for markers of cell growth (Ki67), activity (CD44/CD69), exhaustion (PD-1), degranula tion (CD107a), IFNγ and TNFα in CD8+ T cells unstimulated or stimulated with PMA (50ng/ml) + ionomycin (500ng/ml) for 4 h in vitro. C) Granzyme B+ CD8 T cells treated as in A-B. Dots were counted by ELISpot. D) Expression of active Notch1 (NIC), the Notch1 selective target SNAP23 and the Notch2 selective target BCAT2 in CD8+ T cells. Data are the mean of 3 independent experiments. E, F) IFNγ and GZB ELISpot data from treated tumors. Data are the mean of two independent experiments each containing 5 tumors. P values were calculated by the Student’s t test. G) Naïve YUMM2.1 melanoma cells were co-cultured with TILs extracted from YUMM2.1 tumors treated with IgG or anti-N1, and APCs at a 5:1 ratio, then treated with 10ug/ml anti-N1 for an additional 12 h. Cells were then harvested for flow cytometry. Data are the % of alive cells in the anti-N1 group normalized to control (IgG), which was set at 1 for all treatments. Data are the mean of three independent experiments. Combo = melanoma + TILs + APCs

Article Snippet: IFNγ and GrzB expression in co-cultures were assessed using Mouse ELISpot Development Module (R&D Systems).

Techniques: Flow Cytometry, Activity Assay, In Vitro, Enzyme-linked Immunospot, Expressing, Cell Culture, Control

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