granzyme b elispot development module  (R&D Systems)

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: Scientific Reports

Article Title: Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine

doi: 10.1038/s41598-018-24762-9

Figure Lengend Snippet: B cell responses and total IgG secretion following immunization with quadrivalent vaccines. BALB/c mice (n = 5/group) were immunized subcutaneously at the base of the tail with PBS alone (control) or 80 μg of quadrivalent VLP. Splenocytes were harvested on day 21 and frequencies of total HCV specific antibody secreting cells (ASCs) in each group (PBS, or vaccine combined with Alum, CFA or Montanide) ( A ) and total IgG ( B ) were determined by B cell ELISpot assay. The y-axis shows the antibody secreting cell (ASC) number/million splenocytes. Individual animals are presented for each group, with the mean value being represented by the horizontal bar.

Article Snippet: For the detection of Granzyme B-secreting cells, a Mouse Granzyme B ELISpot Development Module together with reagents from an ELISpot Blue Color Module (R&D Systems, Minneapolis, USA) including a Granzyme B positive control were used according to the manufacturer’s instructions.

Techniques: Vaccines, Control, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine

doi: 10.1038/s41598-018-24762-9

Figure Lengend Snippet: IFNγ T cell responses following immunization with quadrivalent vaccines. ( A ) BALB/c mice (n = 5/group) were immunized subcutaneously at the base of the tail with the quadrivalent VLP combinations as shown. Frequencies of IFNγ secreting cells before and after restimulation were determined by ELISpot assay. ( B ) HHD mice were vaccinated with OT2 Pam2Cys GIL, HCV VLPs or PBS and frequencies of IFNγ secreting cells were determined by ELISpot after stimulation with GILGFVFTL, HCV VLP, HCVcore 132_−140 DLMGYIPLV (DLM), HCVcore 35–44 YLLPRRGPRL (YLL) or the irrelevant HCV NS5B 2594–2602 peptide. ( C ) T cells from BALB/c mice vaccinated with quadrivalent VLP alone were further purified from spleen using Miltenyi Biotec columns and CD4 (L3T4) and CD8a (Ly-2) microbeads and analysed by ELISpot assay. The mean value and standard deviation is shown for each treatment group.

Article Snippet: For the detection of Granzyme B-secreting cells, a Mouse Granzyme B ELISpot Development Module together with reagents from an ELISpot Blue Color Module (R&D Systems, Minneapolis, USA) including a Granzyme B positive control were used according to the manufacturer’s instructions.

Techniques: Vaccines, Enzyme-linked Immunospot, Purification, Standard Deviation

Journal: Scientific Reports

Article Title: Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine

doi: 10.1038/s41598-018-24762-9

Figure Lengend Snippet: Granzyme B responses following immunization with quadrivalent vaccines. BALB/c mice (n = 5/group) were immunized subcutaneously at the base of the tail with the quadrivalent VLP combinations as shown. Splenocytes were harvested on day 21 and frequencies of Granzyme ( B) responses before and after restimulation were determined by ELISpot assay. The mean value and standard deviation is shown for each treatment group.

Article Snippet: For the detection of Granzyme B-secreting cells, a Mouse Granzyme B ELISpot Development Module together with reagents from an ELISpot Blue Color Module (R&D Systems, Minneapolis, USA) including a Granzyme B positive control were used according to the manufacturer’s instructions.

Techniques: Vaccines, Enzyme-linked Immunospot, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TIM-3: A Novel Regulatory Molecule of Alloimmune Activation

doi: 10.4049/jimmunol.0903435

Figure Lengend Snippet: Targeting TIM-3 molecule in WT recipients of a fully MHC-mismatched vascularized cardiac allograft. A, RMT3-23 increases the frequency of IFN-γ–producing, IL-6–producing, IL-17–producing, and granzyme B-producing alloreactive splenocytes compared with that in controls (ELISPOT; spot numbers per a total number of 500.000 responder splenocytes on day 4 posttransplantation). B, Splenocytes from RMT3-23–treated or control animals were separated into MHC class II (MHC-II)–positive (= APC) and MHC-II–negative (= non-APC) cells using appropriate MACS microbeads. Unselected, MHC-II+ and MHC-II− splenocytes were used as responder cells in an IL-6 ELISPOT as described earlier. MHC-II− (non-APC) splenocytes are the main source of RMT3-23–enhanced IL-6 production. C, CD4+ and CD8+ T cells from RMT3-23–treated and control animals were isolated using appropriate MACS microbeads and used as responder cells in an IL-6 ELISPOT as described earlier. CD4+ but not CD8+ T cells from RMT3-23–treated animals show significantly increased frequency of IL-6–producing cells compared with that in control animals. Data are representative of three or more independent experiments using at least n = 3 mice per group. All measurements were done in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001. D, Intracellular staining of graft-infiltrating cells (flow cytometry, live lymphocyte gate). Grafts from RMT3-23–treated recipients show increased frequencies of IL-17+ lymphocytes compared with that in untreated controls. Data are representative of two experiments using n = 3 mice per group. *p < 0.05.

Article Snippet: ELISPOT assays were used to assess the frequency of alloreactive IFN-γ–producing, IL-6–producing, IL-17–producing, and granzyme B-producing cells according to manufacturers’ instructions (ELISPOT Kits for IFN-γ and IL-6 from BD Bioscience; ELISPOT Development Module for IL-17 and Granzyme B from R&D Systems).

Techniques: Enzyme-linked Immunospot, Isolation, Staining, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TIM-3: A Novel Regulatory Molecule of Alloimmune Activation

doi: 10.4049/jimmunol.0903435

Figure Lengend Snippet: TIM-3 blockade in CD28-deficient recipients of a fully MHC-mismatched vascularized cardiac allograft. A, Survival of BALB/c allografts in CD28−/− B6 recipients is abrogated by RMT3-23 (n = 6/6; p = 0.002). Anti–IL-6 mAb can neutralize the effects of RMT3-23 in a dose-dependent manner (no effect with lower doses of anti–IL-6 mAb [n = 6]; complete reversal of accelerated allograft rejection after TIM-3 blockade with higher doses of anti–IL-6 mAb [n = 6]). B, Frozen section of cardiac allografts (day 10 posttransplantation) were stained for FoxP3 and CD3 by immunohistochemistry. FoxP3/CD3 ratio of graft-infiltrating T cells is decreased in RMT3-23–treated animals compared with that in controls (n = 6/6). C, Splenocytes from RMT3-23–treated animals (day 10 posttransplantation) show decreased percentages of CD4+CD25+FoxP3+ Tregs and a trend toward higher CD4+ CD44highCD62Llow effector T cells. D, Splenocytes from RMT3-23–treated animals show increased frequencies of IFN-γ–producing, IL-6–producing, IL-17–producing, and granzyme B-producing alloreactive splenocytes as assessed by ELISPOT (data are shown as spot numbers per a total number of 500.000 responder cells on day 10 posttransplantation). Data are representative of three or more independent experiments using at least n = 3 mice per group. All ELISPOT measurements were done in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: ELISPOT assays were used to assess the frequency of alloreactive IFN-γ–producing, IL-6–producing, IL-17–producing, and granzyme B-producing cells according to manufacturers’ instructions (ELISPOT Kits for IFN-γ and IL-6 from BD Bioscience; ELISPOT Development Module for IL-17 and Granzyme B from R&D Systems).

Techniques: Staining, Immunohistochemistry, Enzyme-linked Immunospot