Wistar rats were immunized subcutaneously (s.c.) and intraperitonially (i.p.) with 50 μg of GST-APEX2 fusion protein dissolved in 500μl PBS, 5 nmol CpG2006 (TIB MOLBIOL) an equal volume of incomplete Freund's adjuvant. Six weeks after immunization a 50 μg boost injection was applied i.p. and s.c. 3 days before fusion. Fusion of the splenic B cells and the myeloma cell line P3 × 63Ag8.653 was performed using polyethylene glycol 1500 according to standard protocols (43 (link)). Hybridoma supernatants were tested by solid-phase enzyme-linked immunoassay (ELISA) using the recombinant GST-fusion protein and verified by Western blotting of whole cell extracts from APEX2 fusions-expressing cell lines (Figure 2B ). Hybridoma cell line from specifically reacting supernatants were cloned twice by limiting dilution. Experiments in this study were performed with clone 20H10 (rat IgG2a/κ).
Cpg2006
Manufactured by TIB Molbiol
Sourced in Germany
CpG2006 is a synthetic oligonucleotide that contains unmethylated CpG dinucleotides. It is a sequence-specific activator of the immune system.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Lps from e coli k12, by InvivoGen (1 mentions)
B cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Monocyte attachment medium, by PromoCell (1 mentions)
1 step ultra tmb elisa, by Thermo Fisher Scientific (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Hybri max, by Merck Group (1 mentions)
Polymyxin b, by Thermo Fisher Scientific (1 mentions)
6 protocols using cpg2006
1
Anti-APEX2 Monoclonal Antibody Generation
2
Isolation and Stimulation of PBMCs
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Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from whole blood or buffy coats (University Hospital Tübingen Transfusion Medicine) using Ficoll density gradient purification, primary B cells from PBMCs using B Cell Isolation Kit II (Miltenyi Biotec, >90% purity by anti-CD19 staining) and hMoMacs using Monocyte attachment Medium (PromoCell). B cells and hMoMacs were stimulated with 200 ng/ml LPS (from E. coli K12, Invivogen) or 2.5 µg/ml CpG 2006 (TIB MOLBIOL) for the indicated time periods. B cells were also stimulated with 2.5 µg/ml CpG 2006 and 5 µg/mL anti-human IgM (Fc5µ, Jackson Immuno Research) for proliferation assays. Carboxyfluorescein-succinimidyl ester (CFSE, Life Technologies) was used to track cell proliferation. Flow cytometry (BD FACSCanto II) was analyzed using FlowJo PC version 10. Further details in Supplemental Information
3
Generation of anti-CXCR5 monoclonal antibodies
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Wistar rats were immunised subcutaneously (s.c.) and intraperitoneally (i.p.) with a mixture of membrane fractions isolated from murine Cxcr5 transfected RBL cells in 500 µl PBS, 5 nmol CpG2006 (TIB MOLBIOL, Berlin, Germany), and 500 µl incomplete Freund’s adjuvant. 6 weeks later, a boost without Freund’s adjuvant was given i.p. and s.c. 3 days before fusion. Fusion of the myeloma cell line P3 × 63-Ag8.653 with the rat immune spleen cells was performed using polyethylene glycol 1500 according to standard procedures62 (link). Supernatants were tested by ELISA for IgG production and analyzed by flow cytometry for specificity using lymphocyte suspensions isolated from WT and Cxcr5−/− mice. Hybridoma cells from supernatants that reacted specifically were subcloned at least twice by limiting dilution. Experiments in this study were performed with clone 6C3 (IgG2a/k).
4
Generation of Monoclonal Antibodies to K15 Protein
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50 μg of the purified GST-K15 (aa 347–489) were injected intraperitoneally (i.p.) and subcutaneously (s.c.) into LOU/C rats using incomplete Freund's adjuvant supplemented with 5 nmol CpG 2006 (TIB MOLBIOL, Berlin, Germany). After six weeks interval a final boost with 50μg K15 and CpG 2006 was given i.p. and s.c. three days before fusion. Fusion of the myeloma cell line P3X63-Ag8.653 with the rat immune spleen cells was performed according to standard procedures. Hybridoma supernatants were tested in a solid-phase immunoassay with K15-GST or GST bound to ELISA plates via mouse anti-GST antibody. Antibodies from tissue culture supernatant recognizing K15 were detected with HRP conjugated mouse mAbs against the rat IgG isotypes (TIB173 IgG2a, TIB174 IgG2b, TIB170 IgG1 all from ATCC, R-2c IgG2c homemade), thus avoiding mAbs of IgM class. HRP was visualized with ready to use TMB (1-Step Ultra TMB-ELISA, Thermo). Monoclonal antibodies (mAbs) that reacted specifically with the K15 were further analysed by western blot and IFA. In experiments, antibodies from clone number 10A6 (IgG G1) and 18E5 (IgG 2b) were used for western blot and immunofluorescence, respectively.
5
Generation of Anti-Fsp1 Monoclonal Antibodies
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Female Wistar rats (RjHan:Wi, age 160 days) were immunized subcutaneously and intraperitoneally with a mixture of 70 µg recombinant C-terminal-His tagged full-length mouse Fsp1 protein in 200 μl PBS, 5 nmol CpG2006 (TIB MOLBIOL), and 200 μl Incomplete Freund’s adjuvant (Sigma-Aldrich). After 8 weeks, a boost without Freund’s adjuvant was given intraperitoneally and subcutaneously 3 days before fusion. Fusion of the myeloma cell line P3X63-Ag8.653 (CRL-1580, ATCC) with the rat immune spleen cells was performed using polyethylene glycol 1500. After fusion, the cells were plated in 96-well plates using RPMI 1640 medium with 20% FBS, glutamine, pyruvate, non-essential amino acids and HAT media supplement (Hybri-Max, Sigma-Aldrich). Hybridoma supernatants were screened 10 days later in a flow cytometry assay for binding to c-His tagged Fsp1 protein captured via biotinylated mouse anti-His antibody (clone HIS 3D5, prepared in-house) to streptavidin beads (PolyAN). Hybridoma supernatant was incubated for 90 min with beads and Atto-488-coupled subclass-specific monoclonal mouse-anti-rat IgG. Antibody binding was analysed using ForeCyt software 8 (Sartorius). Positive supernatants were further validated by Western blotting. Selected hybridoma cells were subcloned by limiting dilution to obtain stable monoclonal cell lines.
6
Optimized Oligomeric Chitosan Protocol
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All chemicals were from Sigma unless otherwise stated. C10-15 chitosan oligomers (a mixture of fragments in an MW range of 2000-3000) were from Carbosynth. Chitin C10-15 was generated from chitosan oligomers using sodium bicarbonate and acetic anhydride acetylation 19 . Purities of >95%, acetylation of >90% were achieved as described in Supplemental Information. Pam3 was from Invivogen, CpG 2006 from TIB Molbiol and, LMP2 and BMLF1 peptides synthesized in house. Polymyxin B was from ThermoFisher.
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