C57 BL/6J mice were immunized with MOG35−55 peptide (Invitrogen) 200 μg, QuilA (Sigma) 20 μg, and keyhole limpet hemocyanin (KLH, Sigma) 20 μg per mouse at day 0. Spleen cells were then isolated at day 10 after immunization. CD4+ T lymphocytes were purified with mouse CD4+ T cell subset column kit (R&D Systems). CD4+ T cells (1 × 106 cells/per well) were co-cultured with DCs at 10:1 (T cells: DCs) and pulsed with MOG 35−55 peptide at 0.1 μM in complete medium with mouse IL-2 at 1 ng/ml for 3 days. Cells were harvested and MOG-primed CD4+ T cells were gated and analyzed by flow cytometry (8 (link), 16 (link)–20 (link)).
Quil a
Manufactured by Merck Group
Sourced in United States
Quil A is a natural adjuvant extracted from the bark of the South American tree Quillaja saponaria. It is used in the formulation of vaccines and other immunological products to enhance immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Mog35 55 peptide, by Thermo Fisher Scientific (2 mentions)
Mouse cd4 t cell subset column kit, by R&D Systems (2 mentions)
Keyhole limpet hemocyanin (klh), by Merck Group (1 mentions)
Pertussis toxin ptx, by Merck Group (1 mentions)
Clonacell hy kit, by STEMCELL (1 mentions)
Enzyme inhibitor cocktail, by Merck Group (1 mentions)
Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ova a5503, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Poly 1 c, by Merck Group (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Incomplete freund s adjuvant ifa, by Merck Group (1 mentions)
Mini beadbeater, by Biospec (1 mentions)
10 protocols using quil a
1
MOG-specific T cell activation protocol
2
Experimental autoimmune encephalomyelitis induction
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The chemicals and drugs used in this study were cx myelin oligodendrocyte glycoprotein peptide human fragment MOG35−55 MEVGWYRPPFSRVVHLYRNGK (Sigma Aldrich, Darmstadt, Germany), saponin from quillaja bark (Quil A; Sigma Aldrich, Darmstadt, Germany), pertussis toxin (PTX; Sigma Aldrich, Darmstadt, Germany), pregabalin [(S)-(+)-3-(aminomethyl)-5-methylhexanoic acid (Zhejiang Chiral Medicine Chemicals Co, Ltd, Hangzhou, China], and acetone (Laboratoires Humeau, La Chapelle-sur-Erdre, France). All drugs were dissolved in injectable sterile 0.9% sodium chloride (NaCl, CDM Lavoisier, Paris, France).
3
Generation of TCRm Monoclonal Antibodies
4
MOG-specific CD4+ T cell generation
5
Preparation and Characterization of Soluble Leishmania Antigen
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The L. major strain (MRHO/IR/75/ER) used in this experiment was previously used for experimental Leishmania vaccine and leishmanin test in Iran (25 , 26 (link)). The method of SLA preparation was carried out using the protocol developed with minor modifications (27 (link)). Briefly, stationary phase promastigotes were harvested and washed four times in HEPES buffer (HS buffer) (10 mM, pH 7.5) containing 10% sucrose. The number of promastigotes was adjusted to 1.2 × 109/ml in buffer solution containing enzyme inhibitor cocktail (50 μl/ml) (Sigma, St. Louis, MO, USA). The parasites were then lysed by freeze-thaw method followed by probe sonication (dr hielscher, Germany) in an ice bath. The supernatant of the centrifuged lysate parasites was collected, dialyzed against HS buffer solution and sterilized by passage through a 0.22 μm membrane and stored at −70°C until use. Quil A was purchased from Sigma Aldrich. Total SLA concentration was determined using BCA (Bicinchoninicacid) protein assay kit (Thermo Scientific, USA) (28 (link)). The antigen was aliquoted and stored at -70°C until use.
6
Intradermal and Subcutaneous Immunizations
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Immunizations were performed by injecting 5 μg QuilA (Sigma-Aldrich, St. Louis, MO, USA) with 50 μg OVA (A5503, Sigma-Aldrich, St. Louis, MO, USA) or E7 peptide-GF001 (RAHYNIVTF, synthesized by Auspep Pty Ltd, (Melbourne, Australia), with purity >80%) in 20 μl PBS into one ear pinnae intradermally or 200 μl PBS subcutaneously.
7
Canine Vaccination for Echinococcus Infection
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The vaccination experiment included nine Beagles. Group 1 was comprised of three dogs that were vaccinated with rEg-TSP11 mixed with the saponin adjuvant Quil A; Group II was comprised of three dogs that were vaccinated with Quil A only; and Group III was comprised of three dogs that were vaccinated with PBS (control group). Each 350 μL dose of vaccine included 200 μg of soluble rEg-TSP11 and 100 μg of Quil A (Sigma) in PBS. Before vaccination, the mixture was stirred overnight at 4°C. All experiments beagle dogs were immunized through subcutaneous injection in the neck and all the dogs were immunized with the same dose four times, at a 14 days interval. Seven days after the last booster vaccination, all nine dogs were challenged orally with 100,000 E. granulosus PSCs. Finally, at 28 days after infection (before eggs appeared), all nine dogs were euthanized and necropsied to collect and count worms as previously described (19 (link), 20 (link)). Thirty worms were chosen randomly from each experimental group and the sizes of developed (≥4 segments) vs. underdeveloped (≤3 segments) worms were determined (21 (link)).
8
Enhancing CPE-Specific Antibody Production
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Solutions containing individual CPE-Ags (10 μg) plus 100 μg Quil A (Sigma-Aldrich, St. Louis, MO, USA. S4521) in 100 μL PBS were injected SC into the backs of the mice at 2 weeks intervals. If CPE-specific IgG Abs were not generated, the mice were injected SC with a newly prepared antigen using the same procedure.
9
Immune Cell Stimulation Assay
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For all experiments, cells were plated one day prior to stimulation. Cells were stimulated at following concentrations (unless mentioned otherwise): AEPS (50 mg/mL), LPS (100 ng/mL, Sigma-Aldrich), poly (I:C) (50 mg/mL, Sigma-Aldrich), Quil A (20 mg/mL), and Alum (200 mg/mL). HEK293T were transfected using Lipofectamineâ 3000 as per manufacturer's instructions (Invitrogen). For NF-kB p65 and STAT3 overexpression, RELA (Myc-DDK-tagged)-pCMV6 or STAT3 (Myc-DDK-tagged)-pCMV6 was transfected to RAW264.7 cells using Lipofectamineâ LTX & PLUS Reagent. After 48 h, cells or culture supernatants were collected for quantitative real-time PCR or ELISA, respectively.
10
OVA Immunization with Adjuvants
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OVA (Sigma, Grave V, A5503) 2mg/mL in PBS was emulsified in Complete Freund's Adjuvant (CFA, Sigma F5881 or Difco 23810, MO USA) or InComplete Freund's Adjuvant (IFA, Sigma F5506, MO USA) using a mini-BeadBeater (Biospec Products, OK-USA) and 100μl injected s.c. at the tail base. In some experiments, 100μg OVA was administered i.p. with 50μg anti CD40 (FGK-45, BioXCell -NH, USA) on day 0, with additional anti-CD40 (i.p.) at day 2, 4, 6, 8. In some experiments, 50μg OVA was mixed with 20μg Quil-A (Sigma) in PBS and injected s.c. at the tail base. For adjuvant-free conditions, OVA was either injected alone or depleted of LPS as described ( 16) and 100μg injected s.c. at the tail base. Where shamimmunized controls were included, PBS and adjuvant was prepared and injected as described for OVA immunizations.
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