Mice were sensitized three times with 10 μg of ovalbumin (OVA) Grade-VI emulsified in 2 mg of aluminum hydroxide (alum) (all from Sigma-Aldrich, St. Louis, MO, USA) by intraperitoneal route with seven-day intervals. Mice were challenged three consecutive days with 30 μg of OVA Grade V (Sigma-Aldrich, St. Louis, MO, USA) by intranasal route seven days after the third sensitization. For challenge, mice were anesthetized with ketamine (100 mg/kg, Sespo Industry and Commerce, Paulínia, SP, Brazil) and xylazine (10 mg/kg, Vetecia Laboratory of Veterinary Products, Jacareí, SP, Brazil).
Ova grade 6
Manufactured by Merck Group
Sourced in United States, Germany
OVA grade VI is a specialized laboratory equipment designed for the precise analysis and processing of biological samples. Its core function is to provide a controlled and consistent environment for the handling and examination of cellular and sub-cellular structures, such as oocytes (eggs) and embryos. The equipment is engineered to maintain specific environmental parameters, ensuring the integrity and viability of the samples under examination.
Automatically generated - may contain errors
Lab products found in correlation
Ova grade 5, by Merck Group (4 mentions)
Imject alum, by Thermo Fisher Scientific (4 mentions)
Ovalbumin (ova), by Merck Group (3 mentions)
Aluminum hydroxide alum, by Merck Group (1 mentions)
6 thioguanine, by Merck Group (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Piperine, by Merck Group (1 mentions)
Recombinant mouse il 4, by BD (1 mentions)
Human il 4, by BD (1 mentions)
Mouse il 4 elisa kit, by BD (1 mentions)
Mouse anti cd3 and anti cd28 antibodies, by BioLegend (1 mentions)
Magnisort mouse cd4 t cell enrichment kit, by Thermo Fisher Scientific (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Inject alum, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Aluminum hydroxide, by Thermo Fisher Scientific (1 mentions)
Budesonide, by AstraZeneca (1 mentions)
Aluminum hydroxide alum, by Thermo Fisher Scientific (1 mentions)
26 protocols using ova grade 6
1
Ovalbumin-Induced Murine Asthma Model
2
Optimizing Immunization with 6-Thioguanine
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Chemicals, including 6-thioguanine, were reagent grade and purchased from Sigma-Aldrich (Saint Louis, MO, USA). 5′ SPO3-CpG oligonucleotide (5′-TCCATGACGTTCCTGACGTT-3′) was purchased from Microsynth (Balgach, Switzerland). Low-endotoxin-grade OVA (<0.01 EU/μg protein), used for immunization, was from Hyglos (Bernried, Germany), and OVA grade VI, used for restimulation, was purchased from Sigma-Aldrich. IL-6 and GM-CSF were purchased from Peprotech (Oak Park, CA, USA).
3
Immunological Assays for Th2 Responses
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Piperine, TMA, dexamethasone, and OVA (Grade VI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Inject Alum was obtained from Pierce Biotechnology (Rockford, IL, USA). The T-PER tissue protein extraction reagent was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Recombinant mouse IL-4 and human IL-4 were purchased from BD Biosciences (San Diego, CA, USA). The mouse IL-4 ELISA kit was purchased from BD Biosciences (San Diego, CA, USA). The MagniSortTM mouse CD4+ T cell enrichment kit was obtained from Invitrogen (Carlsbad, CA, USA). Antibodies specific for STAT6, β-actin, horseradish peroxidase (HRP)-conjugated goat anti-rabbit, anti-mouse antibodies were purchased from Santa Cruz (CA, USA). Antibodies for mouse phospho-STAT6 and CCR3 were obtained from Abcam (Cambridge, UK) and human phospho-STAT6 was purchased from Cell Signaling (Danvers, MA, USA). Mouse anti-CD3 and anti-CD28 antibodies were purchased from BioLegend (San Diego, CA, USA). The RNeasy® Mini Kit was purchased from Qiagen (Valencia, CA, USA).
4
Pneumococcal Infection in Allergic Airway Disease
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WT and IL-22-KO mice were sensitized three times, at 7-day intervals, with 10 µg of OVA Grade VI (Sigma-Aldrich, St. Louis, MO, USA, A2512) and aluminum hydroxide (2 mg) (Thermo Scientific, Waltham, MA, USA, 77161) intraperitoneally. On days 21, 22 and 23 mice were challenged with 30 µg OVA Grade V (Sigma, A5503), intranasally, and samples were collected 24 h after the last challenge. TIGR4 strain of S. pneumoniae (ATCC BAA-334) frozen at −80 °C in tryptic soy broth (TSB) (BD, cat. 211825) containing 10% glycerol was thawed, plated on blood agar and incubated overnight at 37 °C, 5% CO2. After growth, colonies were inoculated in TSB medium, when the growth was checked at OD 600 = 0.06, following incubation for 4 to 5 h. When OD 600 = 0.3, bacteria was counted, and the concentration was adjusted to 50 × 108/mL of colony-forming unit (CFU) in sterile Phosphate Buffered Saline (PBS). Mice were infected with 1 × 108 CFU of S. pneumoniae by intranasal route (20 µL/animal) before the second challenge with OVA. The euthanasia of the animals was performed 48 h after the infection.
5
Murine Model of Allergic Asthma
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Mice were sensitised to OVA by three intraperitoneal (i.p.) injections of 10 µg OVA grade VI (Sigma, Hamburg, Germany) adsorbed to 1.5 mg Al(OH)3 (Pierce, Rockford, IL, USA) diluted in 200 µl phosphate-buffered saline (PBS). Mice were challenged with OVA (grade V) aerosol (1% wt/vol in PBS) twice a week on 2 consecutive days over a period of up to 18 weeks, control groups received PBS. One group of mice was additionally treated with 50 µl of 200 µg/ml budesonide (Astra Zeneca, Lund, Sweden) intranasally (i.n.) 4 hours before each OVA challenge. Group composition is illustrated in Figure 1 . As no differences were detected between control mice in different time points, data is shown only for one PBS group. In an acute model of experimental asthma, OVA sensitised mice were challenged 3 times by OVA aerosol (Figure S2 ). All experiments were performed once with a group size of 6–8 mice treated in parallel in accordance to German animal ethic regulations. Each group was coded and analysed by an investigator blinded to the experimental conditions.
6
Ova-induced Ear Swelling Assay
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Seven days after activated DC transfer, mice were challenged by s.c. injection of 50 μg ova (Grade VI; Sigma‐Aldrich) in 20 μL PBS into the right ear pinna while 20 μL PBS without ova were injected into the left ear pinna for control purposes. Ear swelling was measured in a blinded fashion before and after 48 h injection with a custom‐built spring‐driven micrometer. ova‐specific ear swelling was calculated as the following: (right ear thickness − left ear thickness)48 h − (right ear thickness − left ear thickness)0 h.
7
Evaluating Nanoparticle Immunomodulation in Allergic Lung Inflammation
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To evaluate the immunomodulatory effect of NPs, a protocol of mild allergic inflammation in the lung was used, as previously described (Figure 1A ).23 Briefly, mice were sensitized by repetitive intraperitoneal injections of 1 μg ovalbumin (OVA) (grade VI; Sigma-Aldrich, St Louis, MO, USA) in PBS adsorbed to 2.5 mg aluminum hydroxide (alum) (Thermo Fisher Scientific, Waltham, MA, USA) on days 0, 7, 14, 28, and 42. Blood samples were taken before and after sensitization. OVA/alum-sensitized mice (S mice), compared to non-sensitized mice (NS mice), were characterized by high titers of OVA-specific immuno-globulin E (8.05±1.64 versus 0.1±0.03 μg/mL). At day 52, mice were intratracheally instilled with 50 μg (1 mg/mL) of SiO2 NPs, or with their correspondent SUP (Figure 1B ). For dose-response studies, S mice were instilled with 12.5, 25, or 50 μg of SiO2 and SiO2-PEG NPs. NP instillation was followed by OVA aerosol challenge for 20 minutes with 1% OVA in PBS delivered by a PARI BOY nebulizer (PARI GmbH, Starnberg, Germany). Lung function, flow cytometry, and confocal analysis were performed on day 53. Bronchoalveolar lavage (BAL), histology, scanning electron microscopy (SEM), and messenger (m)RNA expression analysis were performed 5 days after OVA challenge (day 57).
8
Tolerance Induction and OVA-Induced Delayed-Type Hypersensitivity
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After transplantation and/or 10 weeks of feeding, mice were fed by gavage with 25 mg of OVA (Grade III; Sigma‐Aldrich) in 200 µl of phosphate‐buffered saline (PBS) or PBS only as a control on days 0, 3, 6, and 8 for tolerance induction. On day 16, mice were immunized by subcutaneous injection of 300 µg of OVA (Grade VI; Sigma‐Aldrich) in 200 µl of PBS emulsified in complete Freud's adjuvant (Sigma‐Aldrich). On day 34, mice were challenged by subcutaneous injection of 50 µg of OVA (Grade VI) in 10 µl of PBS into the right ear and PBS only into the left ear. Ear swelling was measured before challenge and 48 h later. The DTH response was calculated as followed: (right ear thickness − left ear thickness)48 h − (right ear thickness − left ear thickness)0 h.
9
Immunization Protocol with CpG-B and OVA
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Chemicals were purchased from Sigma-Aldrich (Buchs, Switzerland). CpG-B 1826 ODN (5′-TCCATGACGTTCCTGACGTT-3′) and 5′SPO3-CpG-B 1826 were purchased from Microsynth (Balgach, Switzerland). Low endotoxin grade ovalbumin (OVA) (<0.01 EU/μg protein) (Hyglos, Regensburg, Germany) was used for immunizations and OVA grade VI (Sigma-Aldrich) was used for restimulations. Ground house dust mites (referred to simply as HDM) was obtained from Greer (Lenoir, NC, USA).
10
Allergic Airway Inflammation Model in Mice
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Allergic airway inflammation was induced as previously described [4 (link)]. Briefly, female BALB/c mice (8 weeks old) were sensitized on days 0 and 14 with 20 μg ovalbumin protein (OVA, grade VI, Sigma-Aldrich Chemie GmbH, Munich, Germany) emulsified in 2 mg alum (Imject™ Alum, ThermoFisher Scientific, IL, USA). Nonallergic control animals (PBS) received equal volumes of PBS on days 0 and 14 instead of OVA. Mice were treated with 20 μg recombinant, intact T. suis proteins or heat-inactivated proteins on days 0, 7, and 14 i.p. or left untreated. Intranasal OVA challenge (50 μg OVA/25 μl PBS) was performed on all groups of mice on days 28 and 29. Where indicated, lung function was assessed on day 30 at the Max Delbrück Center for Molecular Medicine (MDC) in Berlin. On day 31, mice were sacrificed, bronchoalveolar lavage (BAL) was performed, and samples were taken for analysis.
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