Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Heat shock protein 70 / MAGE-1 tumor vaccine can enhance the potency of MAGE-1–specific cellular immune responses in vivo

doi: 10.1007/s00262-004-0536-6

Figure Lengend Snippet: IL-2 secretions of splenocytes restimulated with irradiated B16-MAGE-1 were measured using ELISA. C57BL/6 mice were vaccinated as described in Fig. 1. The splenocytes were pooled 2 weeks after the booster. Splenocytes (4×106) were cocultured with 5×105 irradiated B16-MAGE-1 cells for 3 days in a final volume of 2 ml, and the supernatants were obtained and the concentrations of IL-2 were determined using murine IL-2 ELISA kits. Statistical analysis by a paired Student’s t-test demonstrated that the IL-2 concentration in supernatants of splenocytes from mice vaccinated with MAGE-1-HSP70 fusion DNA was higher than that from mice with MAGE-1 DNA (p<0.05, n=6), or with a mixture of MAGE-1 DNA and HSP70 DNA (p<0.05, n=6)

Article Snippet: Cytokine detections Splenocytes (4×10 6 ) were harvested 2 weeks after the last vaccination and cocultured with 5×10 5 irradiated B16-MAGE-1 cells in a total volume of 2 ml of DMEM, supplemented with 10% (v/v) fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin in 24-well tissue culture plates for 72 h. The supernatants were harvested and assayed for the presence of IL-2 using murine IL-2 ELISA kits (Diaclone, Besancone, France) according to the manufacturer’s protocol.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Heat shock protein 70 / MAGE-1 tumor vaccine can enhance the potency of MAGE-1–specific cellular immune responses in vivo

doi: 10.1007/s00262-004-0536-6

Figure Lengend Snippet: MAGE-1–specifc antibody titers in C57BL/6 mice vaccinated with various recombinant DNA vaccines. C57BL/6 mice were vaccinated as described in Fig. 1. Serum samples were obtained from vaccinated mice 2 weeks after the boost vaccination. The presence of MAGE-1–specific antibody was examined by ELISA. The anti-MAGE-1 sera from mice vaccinated with MAGE-1 protein were used as positive controls. The results of the 1:50 dilution are presented showing the mean absorbance (A 450nm)±SE. No anti-MAGE-1 antibodies could be detected in the sera of mice of any vaccinated group

Article Snippet: Cytokine detections Splenocytes (4×10 6 ) were harvested 2 weeks after the last vaccination and cocultured with 5×10 5 irradiated B16-MAGE-1 cells in a total volume of 2 ml of DMEM, supplemented with 10% (v/v) fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin in 24-well tissue culture plates for 72 h. The supernatants were harvested and assayed for the presence of IL-2 using murine IL-2 ELISA kits (Diaclone, Besancone, France) according to the manufacturer’s protocol.

Techniques: Recombinant, Vaccines, Enzyme-linked Immunosorbent Assay

Journal: NPJ Vaccines

Article Title: Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design

doi: 10.1038/s41541-023-00798-5

Figure Lengend Snippet: Mice were immunized (priming) with chimeric proteins either alone (pO, pNO), or bound to T5 CLP (pO-CLP, pNO-CLP) or supplemented with CFA. Mice were subsequently boosted under the same conditions (except that CFA was replaced by incomplete Freund adjuvant, IFA). The interval between priming and boosting was of two ( a ) or six months ( b ). a , b IFNγ-producing splenocytes were quantified by ELISPOT and ( c ) IFNγ production by splenocytes was measured by ELISA after in vitro restimulation with Ova 257-264 peptide 10 days after boosting. The bars correspond to the mean of each group ( n = 5–6) and the circles to results of individual mice. Results were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test with p < 0.05 considered significant (ns non-significant; ** p < 0.01; *** p < 0.001).

Article Snippet: For ELISPOT, plates were revealed with supplied reagents (murine IFNγ ELISPOT kit, Diaclone) and spots were counted with the ImmunoSpot® S6 FluoroSpot Line Plate Reader (C.T.L.).

Techniques: Adjuvant, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, In Vitro, Comparison

Journal: Immunology and cell biology

Article Title: Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells

doi: 10.1038/icb.2017.26

Figure Lengend Snippet: Il17rc transcript abundance is reduced in (A) ST2 cells with shRNA FSTL-1 suppression as well as (B) primary BMSCs from FSTL-1 KO mice when compared to controls. Surface expression of IL-17RC is reduced as measured by FACS determined by (C) %IL17RC+ and (D) mean fluorescence intensity as noted by (E) representative histograms of isotype (dark grey), FSTL-1 KO (light grey) and WT BMSCs (dashed line). *p<0.05, **p<0.01.

Article Snippet: Murine Il17rc (Addgene 46864), Fstl1 (Origene MR204305) and empty vector pCMV6-Entry control (Origene PS 100001) plasmids were transformed in and grown in Mix and Go E. coli (Zymo Research) in 5ml Luria-Bertani broth containing selection antibiotic and prepared using the QIAprep Spin Miniprep kit (Qiagen 27106), per manufacturer’s instructions.

Techniques: shRNA, Expressing, Fluorescence

Journal: Immunology and cell biology

Article Title: Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells

doi: 10.1038/icb.2017.26

Figure Lengend Snippet: FSTL-1 KO BMSC transfection with pCMV- il17rc rescues Il17rc transcript abundance (A). pCMV- Il17rc complementation in FSTL-1 KO BMSCs rescues IL-17A stimulated transcript levels of (B) Il6 and (C) Csf3 . * p<0.05, ** p<0.01, ** p<0.001

Article Snippet: Murine Il17rc (Addgene 46864), Fstl1 (Origene MR204305) and empty vector pCMV6-Entry control (Origene PS 100001) plasmids were transformed in and grown in Mix and Go E. coli (Zymo Research) in 5ml Luria-Bertani broth containing selection antibiotic and prepared using the QIAprep Spin Miniprep kit (Qiagen 27106), per manufacturer’s instructions.

Techniques: Transfection

Journal: Immunology and cell biology

Article Title: Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells

doi: 10.1038/icb.2017.26

Figure Lengend Snippet: FSTL-1 KO BMSC transfection with pCMV- fstl1 rescues (A) Fstl1 and (B) Il17rc transcript abundance. pCMV- Fstl1 complementation in FSTL-1 KO BMSCs rescues IL-17A/TNF stimulated transcript levels of (C) Il6 and (D) Csf3 . *p<0.05, ** p<0.01

Article Snippet: Murine Il17rc (Addgene 46864), Fstl1 (Origene MR204305) and empty vector pCMV6-Entry control (Origene PS 100001) plasmids were transformed in and grown in Mix and Go E. coli (Zymo Research) in 5ml Luria-Bertani broth containing selection antibiotic and prepared using the QIAprep Spin Miniprep kit (Qiagen 27106), per manufacturer’s instructions.

Techniques: Transfection

Journal: Immunology and cell biology

Article Title: Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells

doi: 10.1038/icb.2017.26

Figure Lengend Snippet: Following ActinomycinD treatment, FSTL-1 KO cells had reduced (A) Fstl1 and (8) Il17rc transcript abundance before ActinomycinD treatment and at 1, 2, 4 and 6 hours post-treatment compared with WT BMSCs. Linear regression of Il17rc transcript abundance (C) at various timepoints following ActinomycinD treatment showed similar slopes for WT and FSTL-1 KO BMSCs (−3.254±1.200 and −2.457±1.429, p=0.6748). Newly synthesized mRNA (5-EU labeled) was similar for (D) Hprt , but reduced for (E) Fstl1 and (F) Il17rc , transcripts in FSTL-1 KO cells compared to WT * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001

Article Snippet: Murine Il17rc (Addgene 46864), Fstl1 (Origene MR204305) and empty vector pCMV6-Entry control (Origene PS 100001) plasmids were transformed in and grown in Mix and Go E. coli (Zymo Research) in 5ml Luria-Bertani broth containing selection antibiotic and prepared using the QIAprep Spin Miniprep kit (Qiagen 27106), per manufacturer’s instructions.

Techniques: Synthesized, Labeling

Journal: Frontiers in Aging Neuroscience

Article Title: Microglial Hyperreactivity Evolved to Immunosuppression in the Hippocampus of a Mouse Model of Accelerated Aging and Alzheimer’s Disease Traits

doi: 10.3389/fnagi.2020.622360

Figure Lengend Snippet: Genes analyzed and TaqMan FAM-labeled probes for real-time qPCR.

Article Snippet: Mouse IL1β Quantikine ELISA Kit (MLB00C) was purchased from R&D Systems (Minneapolis, MI, United States), Murine IL6 ELISA Set (861.020.005) from Diaclone (Besançon, France) and Mouse TNFα ELISA Ready-SET-Go (88-7324-22) from Thermo Fisher.

Techniques: Binding Assay

Journal: Frontiers in Aging Neuroscience

Article Title: Microglial Hyperreactivity Evolved to Immunosuppression in the Hippocampus of a Mouse Model of Accelerated Aging and Alzheimer’s Disease Traits

doi: 10.3389/fnagi.2020.622360

Figure Lengend Snippet: Hippocampus of SAMP8 mice revealed an exacerbated acute inflammatory response to lipopolysaccharide (LPS) at young age and null responsiveness at old age. Sickness behavior 3 h after 3 mg/kg i.p. of LPS showed the effect of treatment and a lowering effect of age in the response of both SAMR1 and SAMP8 mice (A) . Blood serum levels of Interleukin 1β (IL1β) were increased in all groups of mice treated with LPS for 3 h regardless of strain and age (B) . Cerebral cortical levels of protein and mRNA of pro-inflammatory cytokines Interleukin 1β (IL1β/Il1b, protein/gene), Interleukin 6 (IL6/Il6, protein/gene) and Tumor necrosis factor α (TNFα/Tnf, protein/gene) were generally elevated by LPS in all mice, with lower protein levels of TNFα in all aged mice and higher mRNA levels of Il1b in SAMP8 mice; mRNA levels of all cytokines in the hippocampus showed exacerbated levels in 6-month-old SAMP8 compared to SAMR1 mice and low levels of cytokines indicative of null responsiveness to a pro-inflammatory injury in 12 month-old SAMP8 mice 3 h after LPS injection (C) . P -values for two-way ANOVA analysis are indicated at the top area of the graph: St, strain main effect; Tr, treatment main effect; Ag, age main effect; and St × Tr × Ag, interaction effect. P -values for Fisher’s LSD post hoc tests are indicated as: * p < 0.05; ** p < 0.01, *** p < 0.001 compared to the corresponding SAMR1 group, the corresponding control treatment group, or the corresponding younger mice group, as indicated. N = 3–10 mice/group.

Article Snippet: Mouse IL1β Quantikine ELISA Kit (MLB00C) was purchased from R&D Systems (Minneapolis, MI, United States), Murine IL6 ELISA Set (861.020.005) from Diaclone (Besançon, France) and Mouse TNFα ELISA Ready-SET-Go (88-7324-22) from Thermo Fisher.

Techniques: Injection, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Microglial Hyperreactivity Evolved to Immunosuppression in the Hippocampus of a Mouse Model of Accelerated Aging and Alzheimer’s Disease Traits

doi: 10.3389/fnagi.2020.622360

Figure Lengend Snippet: Mixed glial cultures of SAMP8 senescent mice had a pro-inflammatory phenotype mainly driven by microglia. Representative images of astrocytes stained with glial fibrillary acidic protein (GFAP) and microglia cultures stained with lectin in mixed glial cultures in control conditions or submitted to a 24-h treatment with lipopolysaccharide (LPS) 100 ng/ml + interferon γ (IFN) 0.1 ng/ml; histograms of the average cell counts of microglia per microscopic field and the percentage of microglia with reactive phenotype shown by globular morphology, as indicated (A) . Histograms of the nitric oxide generation, and the levels of pro-inflammatory cytokines Interleukin 1β (IL1β), Interleukin 6 (IL6) and Tumor necrosis factor α (TNFα) released into the culture media of mixed cultures and microglia cultures, in control conditions or treated with LPS 100 ng/ml + IFN 0.1 ng/ml, as indicated (B) . P -values for two-way ANOVA analysis are indicated at the top area of the graph: St, strain main effect; Tr, treatment main effect; and St × Tr, interaction effect. P -values for Fisher’s LSD post hoc tests between the groups indicated in the graph are as follows: ** p < 0.01, *** p < 0.001. N = 6–10 (microglia micrographs) from 3 independent cultures per group for analysis of microglia number and phenotype in mixed glial cultures, N = 18–24 (mixed glia)/15–26 (microglia) from 4 to 6 independent cultures per group for nitrite determination and N = 4–8 (mixed glia)/3–6 (microglia) independent cultures per group for cytokine analysis. Scale bar = 50 μm.

Article Snippet: Mouse IL1β Quantikine ELISA Kit (MLB00C) was purchased from R&D Systems (Minneapolis, MI, United States), Murine IL6 ELISA Set (861.020.005) from Diaclone (Besançon, France) and Mouse TNFα ELISA Ready-SET-Go (88-7324-22) from Thermo Fisher.

Techniques: Staining, Control