For these studies six to eight week-old female C57BL/6 mice (Jackson Laboratories) were used. Chicken Ova (Sigma) was used as a model protein antigen. Carboxylate-modified fluorescent polystyrene nanoparticles (20 nm and 40 nm, Invitrogen), were used as a model particulate antigen, as well as Ova antigen carriers for immunizations. For immunization experiments 20 nm NPs were conjugated to Ova and every batch of conjugated NPs was analyzed by dot-blot as described previously [17] (link). Biotinylated rabbit anti-Ova antibodies (Thermo Scientific) in combination with streptavidin-FITC (eBioscience) were used to detect Ova antigen and NP-Ova. To highlight the tissue architecture in tissue cryosections, actin-binding Phalloidin-Alexa350 (Invitrogen) was used. Tissue staining with biotinylated Lyve-1 (eBioscience) and E-cadherin (BD Biosciences) antibodies, followed by FITC-conjugated streptavidin (eBioscience) was done in order to visualize the lymphatics and the FRT epithelium. Some tissue sections were stained with antibodies specific for CD11c+ DCs (eBioscience). All antibodies were used at a 1∶100 dilution in blocking buffer.
Chicken ova
Manufactured by Merck Group
Sourced in United States
Chicken OVA is a laboratory product designed for use in research and scientific applications. It serves as a source of chicken eggs, which are commonly used in various experimental and analytical procedures. The core function of Chicken OVA is to provide a standardized and consistent supply of chicken eggs for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated rabbit anti ova antibodies, by Thermo Fisher Scientific (3 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (3 mentions)
Lyve 1, by Thermo Fisher Scientific (2 mentions)
Streptavidin fitc, by Thermo Fisher Scientific (2 mentions)
C57bl 6j male mice, by Charles River Laboratories (2 mentions)
Detoxi gel, by Thermo Fisher Scientific (2 mentions)
Zetapor dispo, by Fujifilm (2 mentions)
Fitc conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
E cadherin, by BD (1 mentions)
Actin binding phalloidin alexa350, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc clone rm4 5, by Thermo Fisher Scientific (1 mentions)
Cd25 apc clone pc61, by BioLegend (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Ovalbumin (ova), by Thermo Fisher Scientific (1 mentions)
Limulus amebocyte lysate assay kit, by Lonza (1 mentions)
Laminarin, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Diff quick staining, by Thermo Fisher Scientific (1 mentions)
Elastase, by Worthington (1 mentions)
Dextran fluorescein, by Thermo Fisher Scientific (1 mentions)
18 protocols using chicken ova
1
Nanoparticle-Ovalbumin Immunization Protocol
2
Olaparib Modulates Allergic Airway Disease
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Mice were sensitized to chicken OVA (Sigma-Aldrich, St. Louis, MO, USA) as described [6 (link)]. The mice were then challenged with aerosolized OVA for 30 min once (single challenge) or once a day for 3 days (multiple challenge). Control groups were not sensitized or challenged. Additional groups of mice received i.p. 1, 5, or 10 mg/kg olaparib (Selleckchem, Pittsburgh, PA, USA) in saline 30 min after OVA challenge. AHR, organ recovery, histopathology, bronchoalveolar lavage (BAL), cytokine and OVA-specific IgE assessment, and FACS analysis were performed as described [6 (link), 22 (link), 23 (link)]. To determine CD4+ T cell populations, spleens were processed to generate single cell suspensions after which splenocytes were stained with antibodies to mouse CD3e (145-2c11-APC) and CD4-FITC (clone RM4-5) (both from e-Bioscience, San Diego, CA, USA). To determine T-regulatory (T-reg) cell populations, splenocytes were stained with CD4 (GK1.5-FITC) and CD25-APC (clone PC61) (from Biolegend, San Diego, CA, USA), and intracellularly with anti-mouse Foxp3 (FJK-16s)-PE (e-Bioscience) followed by FACS analysis. The multiplex assay and FACS were conducted at the LSUHSC Comprehensive Alcohol Research Center Core.
3
Mouse Model of Allergic Asthma
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C57BL/6 J male mice were provided by Vital River Laboratories (Beijing, China). Mice were allowed tap water and rodent chow and were maintained on a 12 h light/dark cycle under a favorable environment (22–24℃). After 1 week of acclimatization, the C57BL/6 mice were randomly divided into two groups (n = 4 per group). The OVA group was sensitized by an i.p. injection (100 µL) of 20 µg chicken OVA (Sigma, United States) emulsified in Imject alum (Pierce, United States) on days 0 and 14 and subsequently challenged for 40 min with an aerosol generated by ultrasonic nebulization of 2% OVA in saline from 24 to 41 days [32 (link)]. The control group was treated with saline in both the sensitization and excitation phases. All experimental procedures used in this study were approved and conducted according to the guidelines of the laboratory Animal Management Committee of Shandong University.
4
Antigen Presentation Assay with OT-II T-Cells
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Antigen presentation assays were carried out, with minor adaptations, as previously described (31 (link)). Maturation of BM-DCs was achieved by an 18 h LPS-treatment at 100 ng/ml followed by an up-regulation of antigen presentation markers such as MHC-II. For specific induction of MHC-II –dependent T-cell activation we employed an OVA-inducible OT-II transgenic T-cell reporter system. Mature BM-DCs were treated with chicken OVA (Sigma) or OVA 323-339 fragment (AnaSpec) for 2 h at 37°C and 5% CO2.
5
Immunization and Infection Protocols
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CFA, HEL, and chicken OVA were purchased from Sigma-Aldrich. For regular CFA immunization, 20 µl of an emulsion of PBS and CFA (1:1) was intradermally injected either in the ear pinna or at the base of the tail of anesthetized mice. Draining auricular or inguinal LNs were harvested at the indicated time points. For immunization with an emulsion of CFA containing HEL-OVA chimeric protein, equimolar amounts of HEL and chicken OVA protein were combined in 0.0235% glutaraldehyde solution in phosphate buffer (pH 7.5) for 1 h at room temperature. After dialysis against PBS, the coupling product was filter-sterilized (0.2 µm) and stored at 4°C. Mice were intradermally injected in the ear pinna with 20 µl of an emulsion of PBS and CFA (1:1) containing 130 µg HEL-OVA conjugate. dLNs and contralateral ndLNs were harvested at the indicated time points. For VSV infection, mice were infected s.c. with 105 PFUs of VSV serotype Indiana dissolved in 10 µl PBS. Draining and nondraining popliteal or auricular LNs were collected at the indicated time points after infection for confocal imaging. All infectious work was performed in designated biosafety level (BSL)-2 and BSL-3 workspaces in accordance with institutional guidelines.
6
Laminarin and OVA Purification
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Laminarin derived from Laminaria digitata was purchased from Invivogen, and chicken OVA was obtained from Sigma-Aldrich. Laminarin and OVA solutions were passed through an endotoxin-remOVAl column (Detoxi-gel: Thermo Fisher Scientific) and subsequently filtered through an endotoxin remOVAl filter (Zetapor Dispo: Wako). The endotoxin levels in the purified Laminarin were evaluated using a Limulus amebocyte lysate (LAL) assay kit (Lonza). OVA peptide 257–264 (SIINFEKL) and OVA 323-339 (ISQAVHAAHAEINEAGR) were purchased from China Peptides (China).
7
Antigen Purification and Peptide Synthesis
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Chicken OVA was obtained from Sigma-Aldrich. OVA solution was incubated with endotoxin removal agarose resin before use. OVA257–264 peptide (SIINFEKL), OVA323–339 (ISQAVHAAHAEINEAGR), TRP2180–188 peptide (SVYDFFVWL), AH1 peptide (SPSYVYHQF) were purchased from Phtdpeptides.
8
Murine Model of Allergic Airway Inflammation
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Mice were sensitized by intraperitoneal injection on days 0 and 7 of 100 μL of 10 mg/mL chicken OVA (Sigma-Aldrich) in phosphate-buffered saline (PBS) emulsified with alum. On days 14, 15, and 16, the mice inhaled aerosolized 1% OVA diluted in PBS for 1 h daily. Matched littermate mice used as controls underwent the same procedures, but with OVA replaced by PBS. The mice were sacrificed 2 days after the final challenge, after which BALF was collected and centrifuged. Cells were spread on slides for Diff-Quick staining (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer protocol in order to analyze the abundance of infiltrated leukocytes, including eosinophils, neutrophils, and macrophages. Lung tissue was fixed for further immunostaining or was lysed in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for subsequent gene-expression or western blot analysis. In some experiments, lung tissue was digested with elastase (Worthington Biochemical Corporation, Lakewood, NJ, USA) to allow the sorting of airway epithelial cells.
9
Intestinal Antigen Uptake Mechanisms
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Six to ten week-old C57BL/6 mice (Jackson laboratories) were used for the studies. Carboxylate-modified fluorescent polystyrene NPs, ranging in size from 20 nm to 2 µm (Invitrogen), and E.coli BioParticles® (Invitrogen) were used as model particulate antigens. Chicken Ova (45 kDa, Sigma), Ova-fluorescein conjugate (Invitrogen), dextran-fluorescein, lysine-fixable dextran-biotin (40 kDa, Invitrogen), and LPS-Alexa Fluor® 488 (3 kDa, Invitrogen) were used as model soluble antigens. Biotinylated rabbit anti-Ova antibodies (Thermo Scientific) and streptavidin-FITC (Biolegend) were used to detect Ova and Ova conjugated to NPs (NP-Ova). Anti-CD11c (eBioscience), Cy-18 (Biolegend) and Lyve-1 (eBioscience) antibodies were used to label LP DCs, goblet cells, and lymphatic ducts respectively. A combination of monoclonal mouse anti-E-cadherin (BD Biosciences) primary antibody and goat anti-mouse-FITC (BD Biosciences) secondary antibody was used to label the IECs. All antibodies were used at a 1∶100 dilution in appropriate blocking buffer. To highlight the tissue architecture in cryosections, actin-binding Phalloidin-Alexa 350 (Invitrogen) was used. DAPI (4′,6-Diamidino-2-Phenylindole, Dilactate, Invitrogen) was used for in vivo labeling of the IEC nuclei. Genistein and chlorpromazine (CPZ) (Sigma) were used for in vivo inhibition of NP uptake at 200–1000 µM and 10–100 µg/ml respectively.
10
OVA-Induced Allergic Asthma in Mice
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We have also constructed OVA models to validate the bioinformatic results. The OVA model was constructed based on the study by Shen et al. (2003) (link). C57BL/6J male mice were purchased from Vital River Laboratories (Beijing, China). Mice were allowed tap water and rodent chow and were maintained at 22°C with a 12 h light-12 h dark cycle. All mice were acclimatized for 1 week before experimentation. The C57BL/6 mice were randomly divided into 2 groups (n = 4 per group). The OVA group was immunized intraperitoneally with OVA and challenged with inhaled OVA. Briefly, mice were injected with 100 µL of 20 µg chicken OVA (Sigma,United States) emulsified in Imject alum (Pierce, United States) on days 0 and 14, and subsequently challenged for 40 min with an aerosol generated by ultrasonic nebulization of 2% OVA in saline from 24 to 41 days. The control group was replaced with saline in both the sensitization and excitation phases. All experimental procedures used in this study were approved and conducted according to the guidelines of the laboratory Animal Management Committee of Shandong University.
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