Six-week-old female BALB/c mice were immunized with 100 μg of purified HCoV-229E S-trimer or S1 protein. Antigens were emulsified in Freund’s complete adjuvant (Sigma-Aldrich; F5881) for the first immunization or Freund’s incomplete adjuvant (Sigma-Aldrich; F5506) for the subsequent boost. Each mouse received three subcutaneous injections at 2-week intervals. Mice with the highest titers of antibodies against the HCoV-229E S-trimer or S1 protein were further boosted by intraperitoneal injection of 200 μg of purified HCoV-229E S-trimer or S1 protein diluted in PBS buffer. Three days after the last injection, spleen cells were collected and fused with SP2/0 cells with PEG1450 (Sigma-Aldrich; P7181) to generate hybridoma cells. Antigen-specific ELISA was used for the hybridoma screening. Positive hybridomas were further subcloned and used for epitope mapping. Finally, ELISA plates were coated with different proteins (the HCoV-229E S-trimer, S1, S1-NTD, and S1-RBD) at 1 μg/ml in CBS (pH 9.6) overnight at 4°C and subsequently blocked and washed. Then, the plates were reacted with the hybridoma culture supernatants at 37°C for 1 h. HRP-conjugated goat anti-mouse IgG (1:5,000 diluted in PBST with 1% [wt/vol] BSA; Boster) was used for detection. Signal reading was carried out in the manner described above. Hybridoma culturing medium was used as a control.
F5506
Manufactured by Merck Group
Sourced in United States, Germany
F5506 is a laboratory equipment product offered by Merck Group. It is a high-performance analytical instrument designed for specific scientific applications. The core function of F5506 is to provide accurate and reliable data analysis, without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
F5881, by Merck Group (28 mentions)
Complete freund s adjuvant, by Merck Group (3 mentions)
T3034, by Merck Group (2 mentions)
Chicken type 2 collagen, by Merck Group (2 mentions)
Polytron pt3100d, by Kinematica (2 mentions)
Female c57bl 6 mice, by Charles River Laboratories (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Peg1450, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg, by Boster Bio (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Rm4 5, by Thermo Fisher Scientific (1 mentions)
Anti cd44 im7, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
High fat diet (hfd), by CLEA Japan (1 mentions)
M3761, by Merck Group (1 mentions)
V2002, by Merck Group (1 mentions)
N1876, by Merck Group (1 mentions)
Methylated bovine serum albumin (mbsa), by Merck Group (1 mentions)
Mycobacterium tuberculosis, by BD (1 mentions)
Acetic acid, by Merck Group (1 mentions)
49 protocols using f5506
1
Generating Monoclonal Antibodies Against HCoV-229E
2
Rabbit Immunization with Viral Antigen
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The purified virus (0.5 ml) was mixed with 0.5 ml of Freund’s complete adjuvant (F5881-Sigma, Germany) It was inoculated subcutaneously into the back of the neck of a male 3–4 months New Zealand Rabbit. Three boosting shots of one-week intervals, and a fourth with a 10-day interval, were inoculated with the virus preparation and Freund’s incomplete adjuvant (F5506-Sigma, Germany). Ten days after the last shot, a blood sample was obtained from the animal’s heart. The serum was separated by centrifugation, aliquoted, and stored alongside the animal’s preimmunization serum at −20°C.
3
Pentasaccharide-CRM197 Immunization in Mice
4
Rabbit Immunization with KLH-Peptide Antigens
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The KLH–peptide complex antigens were dissolved in saline at 2 mg/ml and filtered through 0.22-μm sterile filters. The antigens (0.5 ml) were emulsified with an equal volume of Freund’s complete adjuvant (F-5881, Sigma, Darmstadt, Germany) and injected intradermally at multiple sites along the backs of male New Zealand white rabbits. Two rabbits were injected with one kind of antigen. Booster doses were made in Freund’s incomplete adjuvant (F-5506, Sigma, Darmstadt, Germany) at 2-week intervals with 500-μg doses of the same antigen, totally two times with intervals of 2 weeks. Rabbit serum was collected 14 days after the second booster injection. Antibody titers were detected by indirect ELISA using the respective antigen as coating antigen and the pre-immunization serum of each rabbit as the negative control.
5
Bacterial Infection and TDM Injection in Zebrafish
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Bacterial infections were performed as described previously.44 (link) In brief, 2 dpf larvae were anesthetized in tricaine and injected with ~50–150 fluorescent bacteria along the trunk into a developmentally undefined peri-notochordal space lying between the somatic muscle layers, allowing the injection bolus to spread along the anterior-posterior length of the fish and establishing a largely localized infection in the avascular trunk.
Microinjection of TDM has been described in previous work. In brief, trehalose 6,6′-dimycolate from Mycobacterium bovis (TDM, Sigma-Aldrich #T3034) was resuspended and stored in 2:1 v/v chloroform:methanol at 1 mg/mL. Prior to use, the liquid was evaporated under vacuum and resuspended in incomplete Freund’s adjuvant (IFA, Sigma-Aldrich #F5506) at 2 mg/mL. Larvae were anesthetized in tricaine and injected with approximately 10–20 nL of TDM/IFA or IFA along the trunk. The droplets coalesce into spheres shortly after injection and remain in place for the duration of the experiment. Larvae were then roused in E3 medium supplemented with PTU and allowed to continue development at 28.5°C.
Microinjection of TDM has been described in previous work. In brief, trehalose 6,6′-dimycolate from Mycobacterium bovis (TDM, Sigma-Aldrich #T3034) was resuspended and stored in 2:1 v/v chloroform:methanol at 1 mg/mL. Prior to use, the liquid was evaporated under vacuum and resuspended in incomplete Freund’s adjuvant (IFA, Sigma-Aldrich #F5506) at 2 mg/mL. Larvae were anesthetized in tricaine and injected with approximately 10–20 nL of TDM/IFA or IFA along the trunk. The droplets coalesce into spheres shortly after injection and remain in place for the duration of the experiment. Larvae were then roused in E3 medium supplemented with PTU and allowed to continue development at 28.5°C.
6
Poseltinib Attenuates Collagen-Induced Arthritis
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All animals were obtained from Charles River Japan (150 ± 10 g, 6 weeks old). The experimental protocols were approved by the animal care and use committee of the Hanmi Research Center and performed in accordance with approved guidelines. Briefly, 7-week-old male Lewis rats were immunized with a collagen emulsion of equal volumes of IFA (F5506, Sigma-Aldrich) and bovine type II collagen (total volume, 0.6 mL) via intradermal injection into the base of the tail. Seven days later, the rats were given a booster immunization of 0.3 ml of collagen emulsion in the same manner. The incidence of arthritis was 79% on day 6 after booster immunization, and the rats were randomized into 4 groups when the average clinical score of each animal was 2.8 (on a scale of 16) (n = 20 in each drug-administered group, n = 8 in the CIA control group). Poseltinib was orally administered once a day for 9 consecutive days at a dose of 0.3, 1, or 3 mg/kg. The arthritis score was determined by grading each paw from 0 to 4 based on erythema, swelling, and flexion of the joint. Body weight was also measured three times per week.
7
Generating Anti-PRL-3 Monoclonal Antibodies
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We developed PRLs mAbs using three different types of immunogen: purified prokaryotic expression protein, prokaryotic plasmid pVAX1-Igκ-PRL-3(K-P3) and peptide cross-linked with KLH (KLH, Keyhole Limpet Hemocyanin). Generation of prokaryotic proteins and procedures of plasmid immunization were reported previously 18 (link). For the peptide immunization, we choose the non-homogenous sequence on PRL-3 (compare with PRL-1 and PRL-2) for the peptide design. The sequence of Peptide 1: PGKVVEDWLSLVKAKFCEAEFDQVHFQPLPPAVVKLSDALC-KLH (77-95AA). The sequence of Peptide 2: RLRFKDPHTHKTRCCVMEFDQVHFQPLPPAVVKLSDALC-KLH (108-124AA). Mixture of two peptide (50 μg of each) with complete Freund's adjuvant (Sigma, F5881) was subcutaneously injected into Balb/c-nude mice. Two booster immunizations of peptide mixture with incomplete Freund's adjuvant (Sigma, F-5506) were taken on 3 weeks later. Tail blood was obtained for the titer examination. Hybridoma clones was developed by fusion of SP2/0 myeloma cells with Balb/c-nude mouse spleen-derived B cells which had high titer.
8
Immunization and Flow Cytometry Analysis
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Wildtype (C57BL/6) and NUP210KO mice were immunized subcutaneously with 50 ug of 1W1K peptide (EAWGALANKAVDKA, Cambridge Research Biochemicals) emulsified in Incomplete Freund's Adjuvant (F5506, Sigma). Eight days after immunization draining LNs were harvested, processed to single cell suspensions and stained with I-Ab1W1K tetramer (NIH tetramer bank) for 2 h at room temperature, followed by anti-CD4 (RM4-5, eBioscience) and anti-CD44 (IM7, BioLegend) for 45 min at 4°C. Data were collected on a FACSCantoII (BD Biosciences) and analyzed with FlowJo (Treestar).
9
Induction of Experimental Autoimmune Encephalomyelitis
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Rats were anesthetized with inhalatory anesthesia (Sevorane), the tail base was shaved, and rats were placed in the ventral decubitus position. Animals were injected subcutaneously at the dorsal base of the tail with 200 µl of an emulsion composed of 50% guinea pig brain homogenate emulsified with complete Freund’s adjuvant. The emulsion was composed of 1 ml brain homogenate, 1 ml incomplete Freund’s adjuvant (F5506, Sigma, St. Louis, Mo., USA) and 2 mg of finely ground Mycobacterium tuberculosis (strain H37 Ra, Difco, Detroit, Mich., USA).
10
Immunization Protocol for Antibody Production
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All animal experiments were conducted in compliance with the protocol reviewed by the Institutional Animal Care and Use Committee of Niigata University and approved by the President of Niigata University. C57BL/6NCr mice were purchased from SLC Japan (Shizuoka, Japan), and were fed a high fat diet (HFD) (CLEA Japan, Tokyo, Japan) or normal chow from 4 to 20 weeks of age. Each KLH-conjugated antigenic peptide (20 µg) and an equal volume of complete or incomplete Freund’s adjuvant (Sigma-Aldrich: F5881 (complete) or F5506 (incomplete)) were emulsified by vortexing. For immunization, mice were injected subcutaneously with the KLH-conjugated peptide at the age of 6 weeks (with complete Freund’s adjuvant), 10 weeks (with incomplete Freund’s adjuvant), and 14 weeks (with incomplete Freund’s adjuvant). Control groups received an equal volume of KLH mixed with complete or incomplete Freund’s adjuvant. Plasma was collected from the tail vein of each mouse for analysis of antibody titers.
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