Female, 8-week-old C57BL/6 mice were purchased from Charles River (Germany). Starting one week before immunization, mice were given either 2% (w/v) Glucose water, 10% (v/v) Ethanol (Roche) and 2% (w/v) Glucose (Sigma), or 150 mM Acetate (Sigma), all feedings were changed every 3 days. For primary NP-CGG immunization, mice were then injected i.p. with 100 µg of NP-CGG (LGC, Middlesex, UK) in 200 µL of Imject Alum (Thermo Scientific) according to manufacturer’s instructions. Fourteen days later, mice were boosted with 100 µg of NP-CGG in 200 µL of alum. For TNP-FICOLL immunizations, mice were injected i.p. with 10 µg of TNP-FICOLL (LGC, Middlesex, UK) in 200 µL of Imject Alum (Thermo Scientific). For in vitro TFH differentiation, B-cell receptor transgenic mice specific for HIV-1 Env protein (b12HL mice, in-house breeding) and T-cell receptor transgenic mice specific for chicken ovalbumin 323–339 in the context of I-Ab (OT2 mice, in‐house breeding) were used.
Imject alum
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, United Kingdom, Germany, France
Imject Alum is a laboratory product used as an adjuvant. It is designed to enhance the immune response to administered antigens.
Automatically generated - may contain errors
Lab products found in correlation
Ovalbumin (ova), by Merck Group (36 mentions)
Peg1500, by Roche (24 mentions)
Hypoxanthine, by Thermo Fisher Scientific (21 mentions)
Aminopterin, by Thermo Fisher Scientific (21 mentions)
Rpmi medium, by Thermo Fisher Scientific (16 mentions)
Np klh, by Biosearch Technologies (14 mentions)
Thymidine selection medium supplement, by Thermo Fisher Scientific (13 mentions)
Ova grade 5, by Merck Group (11 mentions)
Hybridoma sfm medium, by Thermo Fisher Scientific (9 mentions)
Np cgg, by Biosearch Technologies (8 mentions)
Ovalbumin (ova), by Thermo Fisher Scientific (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (7 mentions)
Sa3800 cell analyzer, by Sony (7 mentions)
Protein g sepharose 4 fast flow, by GE Healthcare (7 mentions)
Np ova, by Biosearch Technologies (7 mentions)
Thymidine, by Thermo Fisher Scientific (7 mentions)
A5503, by Merck Group (6 mentions)
Tamoxifen, by Merck Group (5 mentions)
Np lps, by Biosearch Technologies (4 mentions)
Fluarix, by GlaxoSmithKline (4 mentions)
338 protocols using imject alum
1
Immunization and in vitro T follicular helper cell differentiation
2
Subcutaneous OVA Immunization in Mice
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Mice were subcutaneously immunized with 100 µg of OVA (Sigma-Aldrich, St. Louis, MO, USA) in the presence of Imject alum (Thermo Scientific, Rockford, IL, USA) in a 1:1 ratio (100 µL of Imject alum to 100 µL of immunogen) and a total volume of 200 µL per animal. Mice were immunized twice with a 1-week interval. One week after the second immunization, blood samples were collected from each individual mouse and serum was kept at −80 °C until used.
3
Peptide Immunization in GIII Mice
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GIII mice were subcutaneously injected with 100 µg of the synthetic peptide in the right footpad. The immunization dose was prepared by dissolving 100 µg of the synthetic peptide in 100 µL of phosphate buffered saline (PBS) (Spitzer et al. 1999 (link)), then adding 100 µL of Imject Alum (an aqueous solution of aluminum hydroxide and magnesium hydroxide plus inactive stabilizers; Thermo Fisher Scientific, USA), and vortexing the solution for 30 min to allow the Imject Alum to effectively adsorb the antigen. A similar booster dose was administered 3 weeks later. The synthetic peptides received by subgroups A–F are summarized in Table 1 . GI mice were subcutaneously injected with two doses of 100 µL of Imject Alum alone with 3-week interval.
Summary of epitope sequences used for the immunized GIII
| Subgroup name | Targeted lymphocyte | Synthesized peptide |
|---|---|---|
| A | CD4 + | NERARELAWATLAAD |
| B | CD4 + | DPAKHAGAIAHLESE |
| C | CD4 + | TQHPVLLRRLIVTKE |
| D | CD8 + | ARELAWATL |
| E | CD8 + | KHAGAIAHL |
| F | CD8 + | LLRRLIVTK |
4
Formulation and Preparation of Adjuvanted Immunogens
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Oil-in-Water formulated TLR-4 agonist (GLA-SE, EM048) was produced by the Infectious Disease Research Institute (IDRI) [26 (link),27 (link)]. EM048 was mixed with recombinant protein in PBS to a final concentration of 50 μg/ml EM048 and 200 μg/ml recombinant protein. Sigma adjuvant system (Sigma) was reconstituted according to the manufacturer’s instructions and combined with recombinant protein to a final concentration of 200 μg/ml recombinant protein. Imject Alum (Thermo Scientific) was mixed with recombinant protein according to the manufacturer’s instructions to a final volume ratio of Imject Alum to immunogen of 1:1 and a final concentration of 200 μg/ml recombinant protein.
5
Immunogenicity of PspA Nanoparticle Vaccine
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Groups of 8–10 CBA/N mice were immunized subcutaneously at the nape of the neck with 0–20 µg PspA, 0–5 µg PspA encapsulated into 50–500 µg polyanhydride nanoparticles, and/or 50 µL of Imject Alum Adjuvant (ThermoFisher Scientific) and delivered in a total volume of 100 µL. Immunization groups for the individual studies (see Tables 1 and 2 ) comprised the following : (i) 25 µg soluble PspA alone, (ii) 25 µg PspA administered with 50 µL Imject Alum, (iii) 5 µg PspA encapsulated into 250 µg 50:50 CPTEG:CPH nanoparticles + 20 µg soluble PspA, (iv) 5 µg PspA encapsulated into 250 µg 20:80 CPH:SA nanoparticles + 20 µg soluble PspA, (v) 1 µg soluble PspA alone, (vi) 1 µg PspA administered with 50 µL Imject Alum, (vii) 1 µg PspA encapsulated into 50 µg 50:50 CPTEG:CPH nanoparticles, (viii) 0.5 µg PspA encapsulated into 25 µg 50:50 CPTEG:CPH nanoparticles + 25 µg blank 50:50 CPTEG:CPH nanoparticles + 0.5 µg soluble PspA, or (ix) saline control. Blood was collected from mice via saphenous vein and serum was isolated following centrifugation (10,000 rcf for 10 min) at days 17 or 21 DPI. Serum was stored at −20°C until analysis could be performed.
6
Ova-Induced Allergic Airway Inflammation
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Mice were sensitized to chicken Ova from Sigma-Aldrich (Oakville, ON, Canada) using the protocol reported by Jacobsen et al.5 (link) with the modifications described here. Briefly, Ova was prepared at 0.909 mg/mL in PBS and mixed as per the manufacturer’s instructions with Imject Alum (Thermo Scientific, Waltham, MA, USA) at a ratio of 11:14 of Ova to Imject Alum. Mice were first sensitized to Ova by IP injection of 100 µL of either PBS or Ova/Imject Alum mix on days 0 and 14. On days 24, 25, and 26, mice were challenged with a 25-min exposure to nebulized 1 mg/mL Ova in saline. For the challenge, mice were housed in a pie cage (Braintree Scientific, Braintree, MA, USA) that received nebulized Ova from a Schuco cup nebulizer, with pressure on the vacuum pump set at <1 kg/cm2. Mice were imaged on day 28.
Acute silica exposure was performed similar to Ferreira et al.60 (link). Briefly, mice were anesthetized with isofluorane and then instilled intranasally with sterile saline alone or sterile saline containing 10 mg crystalline silica with a particle size of 0.5–10 μm (Sigma Chemical, St Louis, MO). Twelve hours later, animals were prepared for lung intravital microscopy as described above.
Acute silica exposure was performed similar to Ferreira et al.60 (link). Briefly, mice were anesthetized with isofluorane and then instilled intranasally with sterile saline alone or sterile saline containing 10 mg crystalline silica with a particle size of 0.5–10 μm (Sigma Chemical, St Louis, MO). Twelve hours later, animals were prepared for lung intravital microscopy as described above.
7
Immunization Protocols for Antibody Responses
8
OVA-Induced Allergic Asthma Protocol
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Six to eight week old male BALB/c mice from The Jackson Laboratory (Bar Harbor, ME, USA) were housed in a specific pathogen free facility. All animal experiments were approved by the University of Washington Institutional Animal Care and Use Committee. Figure 1 depicts the OVA sensitization and challenge protocol. Briefly, mice were sensitized at day −28 by IP administration of 1 μg of OVA (grade V; Sigma, St Louis, MO, USA) at 1 μg/μL in normal saline, mixed 1:1 with the adjuvant Imject Alum (Thermo Scientific Pierce, Rockford, IL, USA). Mice were resensitized by IP route on day −15, followed by EC route on day −14. For EC sensitization, mice were anesthetized with isoflurane and shaven. The mid-back was gently scratched ten times, and a 1.5 cm × 1.5 cm gauze patch impregnated with 1 mg of OVA at 10 mg/mL in dH2O was affixed to the scratched site under Tegaderm (3M Health Care, St Paul, MN, USA) and Elastikon (Johnson & Johnson Consumer Co, Inc, Skillman, NJ, USA). The patch was left intact for 7 days. Mice were challenged on day 0 by repeated daily EC application until harvest. Sham-treated mice received normal saline by IP route and dH2O by EC route.
9
Ovalbumin-Induced Airway Inflammation in Balb/c Mice
10
Mouse Model of House Dust Mite Allergy
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Mice were sensitized intraperitoneally on days 0 and 14 with or without 200μl of a 12.5% solution of Imject alum (Fisher Scientific, Edmonton, AB, Canada) in saline, either alone or with 1μg of rDer p 2 or the equivalent amount of Der p 2 delivered as BP-Der p 2 or present in a commercial house dust mite (HDM) extract (Stallergenes Greer, Lenoir, NC, USA). The quantity of Der p 2 in BP-Der p 2 and in the HDM extract was quantified by western blot. In HDM extract, 1μg Der p 2 was present in 41μg total HDM protein. Starting on day 21, all mice were lightly anaesthetized with isoflurane and challenged intranasally with 41μg total HDM protein in a 30μl volume on four consecutive days. After 24 hours, airway hyperresponsiveness (AHR) was assessed (see below), and bronchoalveolar lavage (BAL) fluid and blood were harvested for ex vivo analysis.
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