EAE was induced in DBA/1j and C57BL/6 mice with either 50 μg of recombinant rat MOG1–125,41 (link) emulsified in incomplete Freund's adjuvant (Sigma-Aldrich) supplemented with 3 mg ml−1 of heat-inactivated Mycobacterium tuberculosis H37RA (Difco Laboratories, Oxford, UK), or in C57BL/6 mice with 100 μg of MOG35–55 peptide (AnaSpec, Fremont, CA, USA) emulsified in incomplete Freund's adjuvant (Sigma-Aldrich) supplemented with 4 mg ml−1 of heat-inactivated Mycobacterium tuberculosis H37RA (Difco Laboratories). The emulsion was injected intradermally at the base of the tail. In some experiments (as indicated in the figure legends), animals were treated with 200 ng of pertussis toxin (Enzo Life Sciences, Farmingdale, NY, USA) via intraperitoneal injection on days 0 and 2. Clinical assessment was performed daily by an observer ‘blinded' to mouse genotype and according to the following criteria: 0, no disease; 1, decreased tail tone; 2, abnormal gait (ataxia) and/or impaired righting reflex (hind limb weakness or partial paralysis); 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, hind limb paralysis with partial forelimb paralysis; and 6, moribund or dead.
Incomplete freund s adjuvant
Manufactured by Merck Group
Sourced in United States, China, Germany, United Kingdom, Sao Tome and Principe, Canada, Australia, Spain, France, Italy, Israel, Macao, Senegal, Japan
Incomplete Freund's adjuvant is a laboratory reagent used to enhance the immune response in certain immunological experiments. It is a water-in-oil emulsion that contains mineral oil and mannide monooleate, but does not contain killed or attenuated microorganisms like the complete Freund's adjuvant. The incomplete Freund's adjuvant is used to induce a strong, sustained immune response without the granulomatous reaction associated with the complete formulation.
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Lab products found in correlation
Complete freund s adjuvant, by Merck Group (446 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (23 mentions)
Pertussis toxin, by Merck Group (21 mentions)
Ovalbumin (ova), by Merck Group (17 mentions)
Bovine serum albumin (bsa), by Merck Group (13 mentions)
Bovine type 2 collagen, by Chondrex (10 mentions)
Pertussis toxin, by List Biological Laboratories (9 mentions)
Mycobacterium tuberculosis h37ra, by BD (9 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (8 mentions)
Complete freund s adjuvant cfa, by Merck Group (6 mentions)
Complete freund adjuvant (cfa), by Merck Group (6 mentions)
Keyhole limpet hemocyanin (klh), by Merck Group (5 mentions)
Bovine type 2 collagen, by Merck Group (5 mentions)
Methylated bovine serum albumin (mbsa), by Merck Group (5 mentions)
Tween 20, by Merck Group (5 mentions)
Nmri mice, by Charles River Laboratories (5 mentions)
Female balb c mice, by Charles River Laboratories (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Polyethylene glycol, by Merck Group (4 mentions)
691 protocols using incomplete freund s adjuvant
1
Experimental Autoimmune Encephalomyelitis Induction
2
Immunization Protocol for Antisera Production
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Female BALB/c mice (8–9 weeks old, n = 7) were purchased from Charles River Laboratories (Wilmington, MA) and used for immunization and collection of specific antisera according to standard procedures as described previously [39 ]. The animals, which were designated as R1–R7, received an initial subcutaneous injection of 0.2 mL of a 1:1 emulsion of 50 μg KLH-Val-CONH2 immunoconjugate in 0.1 mL of PBS (pH 7.4):0.1 mL of complete Freund´s adjuvant (Sigma–Aldrich, USA). Three subcutaneous booster injections consisting of 0.2 mL of a 1:1 emulsion of 50 μg KLH-Val-CONH2 immunoconjugate in 0.1 mL of PBS (pH 7.4):0.1 mL of incomplete Freund’s adjuvant (Sigma–Aldrich, USA) were administered, followed by a final intraperitoneal booster injection of KLH-Val-CONH2 in 0.1 mL of PBS (pH 7.4):0.1 mL of incomplete Freund’s adjuvant 30 days after the previous immunization. Blood (50 μL) was collected from the tail vein of the mice every 2 weeks after each immunization and was centrifuged in an Eppendorf 5804R centrifuge at 1200 rpm for 10 min at 4 °C. The collected sera were stored at −20 °C until further use.
3
Generating Anti-Chi3l1 Monoclonal Antibodies
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Two female New Zealand White rabbits (10–16 weeks of age, Charles River Labs) were injected subcutaneously with 400 μg of hChi3l1 and Complete Freund’s Adjuvant (Sigma). The rabbits were boosted with 200 μg of hChi3l1 and inComplete Freund’s Adjuvant (Sigma) subcutaneously. To generate anti-mChi3l1 antibodies, the rabbit-#2 was boosted with mChi3l1 (200 μg) and inComplete Freund’s Adjuvant (Sigma) subcutaneously.
The peripheral blood mononuclear cells (PBMCs) were isolated from the immunized rabbit by the ACCUSPIN™ system (Sigma). The antigen-specific memory B cells were enriched by biotinylated Chi3l1 and Streptavidin MicroBeads (Miltenyi Biotec) according to the manufactory’s instructions. The single memory B cells were seeded into 96-well cell culture plates, at a density of about one cell/per well, with pre-coated EL4-B5 feeder cells (Kerafast). The feeder cells were irradiated with 50 Gy in a gamma radiation chamber before use. The single memory B cells were cultured in 100 μL of RPMI-1640 (10% FBS) with IL-2 (10 U/mL, R&D Systems), IL-21 (10 U/mL, R&D Systems) at 37 °C, 5% CO2 and 93% humidity for 14 days. The supernatant was collected to detect the antibody concentration and the antigen-binding of the antibodies.
The peripheral blood mononuclear cells (PBMCs) were isolated from the immunized rabbit by the ACCUSPIN™ system (Sigma). The antigen-specific memory B cells were enriched by biotinylated Chi3l1 and Streptavidin MicroBeads (Miltenyi Biotec) according to the manufactory’s instructions. The single memory B cells were seeded into 96-well cell culture plates, at a density of about one cell/per well, with pre-coated EL4-B5 feeder cells (Kerafast). The feeder cells were irradiated with 50 Gy in a gamma radiation chamber before use. The single memory B cells were cultured in 100 μL of RPMI-1640 (10% FBS) with IL-2 (10 U/mL, R&D Systems), IL-21 (10 U/mL, R&D Systems) at 37 °C, 5% CO2 and 93% humidity for 14 days. The supernatant was collected to detect the antibody concentration and the antigen-binding of the antibodies.
4
Experimental Autoimmune Encephalomyelitis Induction
5
Transgenic Mice Immunized with NY-ESO-1 Peptide
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Transgenic mice expressing the human HLA-A*0201 gene were obtained from Jackson Lab and housed in specific pathogen free (SPF) mouse facilities. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the Institute of Microbiology, Chinese Academy of Science (IMCAS), conducted as previously described, and in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of IMCAS Ethics Committee. Briefly, 8- to 12-week old mice were immunized subcutaneously with 100 μg of HLA-A*0201-restricted peptide NY-ESO-1157−165 (SLLMWITQC) or an equal volume of PBS as control, plus 120 μg of HBVc 128-140 helper peptide, emulsified in 100 μl of incomplete Freund's adjuvant (Sigma, Saint Louis, Missouri), as described before (34 (link), 49 (link)). A booster immunization was given using complete Freund's adjuvant (Sigma, Saint Louis, Missouri) instead of incomplete Freund's adjuvant 1 week later. One week after the booster immunization, mice were euthanized, and splenocytes were harvested and cultured.
6
Protective Immunity Against S. suis
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Immunization of mice and challenge assays with pathogenic S. suis strains were conducted as previously described (Li et al. 2007 ). Six-week-old female BALB/c mice were randomly divided into five groups with 12 in each. Purified SsPrsA (100 μg) emulsified with incomplete Freund's adjuvant (Sigma) was inoculated subcutaneously for group I and III mice and those of groups II and IV with incomplete Freund's adjuvant only. Group V mice served as a control without any injection. A booster immunization was conducted after two weeks. Ten days after the second inoculation, mice were challenged intraperitoneally with 8.0 × 10 8 CFU of the S. suis serotype 2 strain HA9801 (SS2-HA9801) in groups I and III and those in groups II and IV with 1.2 × 10 8 CFU of the S. suis serotype 9 strain JX13 (SS9-JX13). The mice were continuously observed twice a day for one week. Pre-immune negative sera were retrieved prior to the first immunization and immune sera were collected after boosting. Antibody responses were determined by indirect ELISA with SsPrsA as the coating antigen. Western blotting was performed as described above to evaluate the cross-reaction of SsPrsA-immunized mice sera with the surface proteins or the whole cell lysate proteins of heterologous strains of SS2 and SS9.
7
Synthetic Saponin Vaccine Evaluation
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The ability of the synthetic saponin vaccine formulations to stimulate in vivo immune responses was investigated by vaccinating 6–8‐week‐old C57BL/6 J mice.
At day −1, mice were adoptively transferred with 8×106 OVA‐specific CD4 and CD8 T cells from congenic OT‐I and OT‐II donors by intravenous injection via the tail vein. At days 0 and 14 groups of mice (n=3) were immunized by subcutaneous injection with a) OVA (10 μg, albumin from chicken egg white, Grade VI, ≥ 98 %, Sigma) in QA ISCOM in phosphate‐buffered saline (PBS, 100 μg saponin), b) OVA (10 μg) in24 (50 %):PC:cholesterol particles in PBS or Freund's incomplete adjuvant (Sigma–Aldrich, USA), c) OVA (10 μg) in 31 (50 %):PC:cholesterol particles in PBS, or d) OVA (10 μg) in Freund's incomplete adjuvant. On day 28, all mice were euthanized, and single‐cell suspensions were then prepared from the harvested lymphoid tissues.
At day −1, mice were adoptively transferred with 8×106 OVA‐specific CD4 and CD8 T cells from congenic OT‐I and OT‐II donors by intravenous injection via the tail vein. At days 0 and 14 groups of mice (n=3) were immunized by subcutaneous injection with a) OVA (10 μg, albumin from chicken egg white, Grade VI, ≥ 98 %, Sigma) in QA ISCOM in phosphate‐buffered saline (PBS, 100 μg saponin), b) OVA (10 μg) in
8
Immunization of Mice with GST-LAE Fusion Proteins
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The animal experimental protocols used were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Bestar Laboratories (S) Pte Ltd. where THT served. Each of the five groups of mice comprised of six 4-week-old female JCR mice were immunized with each of the respective gel-purified GST-LAE fusion proteins (LAE1, LAE3, LAE5 and LAE7) or the negative control GST protein. On day 0, each mouse was injected subcutaneously with 100 μL of antigen containing 25 μg of the GST-LAE fusion protein mixed with an equal volume of Freund’s incomplete adjuvant (Sigma -Aldrich, St. Louis, MO, USA). On days 14 and 28, the mice were bled and boosted with the same antigens mixed with an equal volume of Freund’s incomplete adjuvant (Sigma). An additional injection of 25 μg of protein antigen without adjuvant was administered at day 42 (without bleeding). Blood samples of 100 μL each were taken from every mouse by facial bleeding on days 14 and 28, and the mice were sacrificed after the final bleeding on day 45.
9
Equine RBD Protein Immunization Protocol
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Ten healthy equines aged 6–10 years were housed under standard breeding conditions after quarantine inspection. Before immunization, 3 mg of RBD protein in 3 ml of PBS was mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich) for priming, 6 mg of RBD protein in 3 ml of PBS was mixed with an equal volume of Freund's incomplete adjuvant (Sigma-Aldrich) for the first boost, and 12 mg of RBD protein in 3 ml of PBS was mixed with an equal volume of Freund's incomplete adjuvant (Sigma-Aldrich) for the second boost. The equines received intramuscular injections on day 0, 12, and 22. Serum samples were collected from the jugular vein on day 7, 19, and 27 to monitor variations in antibody responses. A large amount of plasma was collected after the third boost, and each collection was conducted 7 days after the boost with 12 mg of RBD.
10
Antibody Generation Against rLdSir2RP Protein
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The purified rLdSir2RP protein was used for raising antibodies in swiss mice. Mice were first immunized using 50 μg of rLdSir2RP in Freund’s complete adjuvant (Merck). After 15 days, the mice was given 3 booster doses of 25μg rLdSir2RP each in incomplete Freund’s adjuvant (Merck) at 2-weeks interval and blood was collected for serum 8 days after the last immunization. Antibody titre was determined by ELISA and was found to be 1:64000. For immunoblotting experiment, purified rLdSir2RP protein and SLD were resolved on 12% SDS—PAGE and transferred on to nitrocellulose membrane using a semi-dry blot apparatus (Amersham) [18 (link)]. After overnight blocking in 5% BSA, the membrane was incubated with antiserum to the rLdSir2RP protein at a dilution of 1:2000 for 120 min at room temperature (RT). The membrane was washed three times with PBS containing 0.5% Tween 20 (PBS-T) and then incubated with goat anti-mice IgG HRP conjugate (Bangalore Genie) at a dilution of 1:10,000 for 1h at RT. Blot was developed by using diaminobenzidinehydrochloride + imidazole +H2O2 (Sigma).
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