The hybridoma was prepared following the manufacturer’s instructions (Roche, Basel, Switzerland) with minor modifications. Briefly, the mouse spleen cells were mixed at a ratio to Sp2/0-Ag14 of 5:1 (ATCC, VA, USA) in a sterile 50-ml conical tube, which was centrifuged to pellet the cells at 800 rpm for 10 minutes. After discarding the supernatant, 1 ml of 50% PEG 1500 (Roche) was slowly added to the cell pellet dropwise over a 1-minute period and the cells were swirled for 90 seconds in a 37 °C water bath. Cell fusion was stopped by adding Roswell Park Memorial Institute medium (RPMI) 1640 (Gibco, CA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, CA, USA) with gentle swirling at RT for 10 minutes. After washing with RPMI 1640 twice, cells were suspended in 30 ml of RPMI1640 supplemented with 10% FBS, 10% BM Condimed H1 (Roche) and 1x HAT (Gibco), plated 2.5 ml per well in a 6-well culture dish and incubated at 37 °C in a 5% CO2 incubator. Limiting dilution was carried out for selection of a single colony, which was amplified in RPMI 1640 supplemented with 10% FBS, 10% BM Condimed H1 (Roche), 1× HT (Gibco) and 1x hybridoma fusion & cloning supplement (Roche). The supernatant was harvested for ELISA of antibody activity to pp65422-439.
Sp2 0 ag14
Manufactured by American Type Culture Collection
Sourced in United States
Sp2/0-Ag14 is a murine myeloma cell line. It is a widely used host cell line for the production of monoclonal antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Complete freund s adjuvant, by Merck Group (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Peg1500, by Roche (1 mentions)
Rpmi 1640, by Roche (1 mentions)
Bm condimed h1, by Roche (1 mentions)
Ni nta kit, by Qiagen (1 mentions)
Fbs and antibiotics, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Clonacell hy hybridoma kit, by STEMCELL (1 mentions)
Aminopterin, by Merck Group (1 mentions)
Incomplete adjuvant, by Merck Group (1 mentions)
Hypoxantine, by Merck Group (1 mentions)
Rpmi 1640 culture medium, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Eurobio Scientific (1 mentions)
Dmem culture medium, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Thymidine, by Merck Group (1 mentions)
Peg 1500, by Merck Group (1 mentions)
Purification kit, by Qiagen (1 mentions)
15 protocols using sp2 0 ag14
1
Hybridoma Production and Antibody Screening
2
Tetherin-Specific Monoclonal Antibodies from H. pylori NAP Immunization
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Tetherin-specific mouse hybridomas were generated following vaccination of mice with Helicobacter pylori neutrophil-activating protein chimera (NAP). NAP chimera proteins significantly enhance immunogenicity of antigens and methods of protein expression, mouse immunization, and hybridoma isolation have been previously described (48 (link)– (link)50 (link)). NAP-tetherin was cloned into the bacterial expression plasmid pET28a+ with His6 and MBP N-terminal tags for purification, separated by a TEV cleavage site. BL21(DE3) E. coli were transformed with the pET28 NAP-tetherin and His-tagged protein was purified with a NiNTA kit (Qiagen). Six- to 8-week-old BALB/c mice were intraperitoneal injected with 50 μg of purified NAP-tetherin with Freund’s adjuvant and boosted with 25 μg additional antigen by intravenous injection. Spleen cells were collected and fused with myeloma line Sp2/0-Ag14 (ATCC) using polyethylene glycol 4000. Hybridomas were screened for tetherin specific reactivity by antigen-mediated ELISA and immunoblotting. Two tetherin specific MAbs were identified, 34F11 and 35H2. Hybridoma supernatant was directly used as primary antibodies for experiments (1:20 to 1:50 dilution).
3
Monoclonal Antibody Production against BLV
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At eight weeks of age, BALB/c mice were immunized intraperitoneally with 0.2 mL (0.5 mg/mL) of recombinant partial BLV gp51 protein emulsified 1 : 1 in Freund's complete adjuvant (Sigma, USA). The mice were boosted three times with the same quantity of antigen at two week intervals. Four days after the last immunization, their splenic mononuclear cells were isolated and fused with murine myeloma cells (SP 2/0 Ag14) (ATCC, USA) using 50% polyethylene glycol (PEG)-1000 (Roche, Germany). The hybridomas were generated by HAT medium selection (Sigma) and screened using a recombinant BLV protein-based enzyme linked immunosorbent assay (ELISA). The positive hybridoma cells were cloned by limiting-dilution, and 18 positive hybridoma lines were obtained. The selected MAbs were further tested for their ability to interfere with ligand-receptor binding in a blocking ELISA, as previously described [28 (link)]. Western blot and isotype analyses were performed as previously described [34 (link)] using the hybridoma with superior blocking ability.
4
Generating Monoclonal Antibodies against Measles Virus
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The mouse myeloma line Sp2/0-Ag14 was purchased from ATCC and grown in the recommended medium. Five-week-old female BALB/c mice were injected with 2 × 106 TCID50 of MV-GFP via the i.p. route. Immunization was repeated twice on days 7 and 21, and anti-MV serum antibody titers were determined by ELISA. Mice with the highest titer received a final intravenous (i.v.) boost with 106 TCID50 MV-GFP. Three days later, spleen cells were harvested and hybridomas were generated by using the Sp2/0-Ag14 hybridoma fusion partner.63 Hybridomas were grown in DMEM (Sigma) supplemented with 10% FBS and antibiotics (Thermo Fisher Scientific). Hybridoma culture supernatants were tested by antigen-mediated ELISA and immunoblotting for MV antigen-specific mAb production. Hybridomas against MV N and P protein were cloned and culture supernatants were collected. mAbs 8A11 (anti-N) and 9E11 (anti-P) isotypes were identified using an IsoStrip monoclonal antibody isotyping kit (Santa Cruz Biotechnology) and used for the experiments. Before the hybridoma generation description in the present study, as part of the complete characterization, the N protein-specific mAb 8A11 was tested in immunoblot and immunohistochemistry for detection of MV infection,40 ,64 (link) including in formalin-fixed and paraffin-embedded clinical tissue samples.
5
Monoclonal BRAA Production Protocol
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Monoclonal BRAA were produced using a ClonaCell®-HY Hybridoma Kit purchased from STEMCELL Technologies (Vancouver, Canada). The protocol used has been described in the manual. Mouse myeloma cells (Sp2/0-Ag14 – ATCC (USA)) were fused with spleen cells from MRL/lpr #2. Cell viability was determined using trypan blue. Visible colonies on the petri dishes were plated in 96-well plates and tested for the presence of BRAA using ELISAs and if positive were transferred to 24-well plates. If the cells were still positive, they were cultured in petri dishes and then stored at −80°C.
6
Monoclonal Antibody Generation from Immunized Mice
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5 Balb/c mice (8–12 weeks of age) per immunogen were immunized by intraperitoneal or foot pad injections using either sGP or GP proteins (total of 20 mice: 5 mice x 2 injection sites x 2 antigens) emulsified in Freund's complete adjuvant (Sigma) (first injection) or incomplete adjuvant (Sigma) (boost injections) (5–10 μg of proteins every 2 weeks). Three days after the last injection, spleen cells (intraperitoneal injections) or popliteal lymph node cells (foot pad injections) were fused with SP2/0 myeloma cells (Sp2/0-Ag14 (ATCC CRL-1581), using polyethylene glycol (PEG 1500) (Sigma). Hybridomas were grown in DMEM culture medium (Sigma) containing 20% Fetal Calf Serum (FCS) (Eurobio, France), penicillin (100 IU/ml) and streptomycin (100 μg/ml) and supplemented with hypoxantine (1×10−4 M), aminopterin (4×10−7 M) and thymidine (1.6×10−5 M) (HAT) (Sigma). After ten to fourteen days of culture, secreting hybrids were identified by analysis of culture supernatants by indirect enzyme-linked immunosorbent assay (ELISA). Hybridomas from selected antibody were cloned by limiting dilution and processed according to conventional methods. Clones secreting antibody of desired reactivity were expanded in 25 and 75 cm2 flasks (Nunc, Denmark), harvested and cryopreserved in 40% FCS, 10% Dimethylsulfoxide (DMSO) (Sigma) and 50% RPMI-1640 culture medium (Sigma).
7
Influenza Virus Cultivation and Plasmid Construction
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Madin-Darby canine kidney (MDCK) cells and HEK 293 T cells were maintained in our lab. SP2/0 mouse myeloma cells were purchased from ATCC (Sp2/0-Ag14; ATCC CRL-1581). All cells were grown in complete Dulbecco’s modified Eagle medium (DMEM; Life Technologies, US) supplemented with 10% foetal bovine serum (FBS; Gibco, US).
Influenza viruses used in this study were mouse-adapted H7N9 (A/Shanghai/2/2013, SH/2/13), H9N2 (A/chicken/Hunan/2/2008, HN/2/08) and H1N1 (A/Puerto Rico/8/1934, PR8) viruses, which were grown in 8–10-day-old embryonated chicken eggs, and titres were determined on MDCK cells in the presence of TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin (Sigma, US).
pCAGGSP7/NA was constructed by cloning the NA gene from the influenza virus strain A/Shanghai/2/2013 (H7N9) into expression vector pCAGGSP7, as described in our previous studies [15–20 (link)]. The plasmid was propagated in Escherichia coli XL1-blue bacteria and purified using Qiagen Purification Kits (Qiagen, US).
Influenza viruses used in this study were mouse-adapted H7N9 (A/Shanghai/2/2013, SH/2/13), H9N2 (A/chicken/Hunan/2/2008, HN/2/08) and H1N1 (A/Puerto Rico/8/1934, PR8) viruses, which were grown in 8–10-day-old embryonated chicken eggs, and titres were determined on MDCK cells in the presence of TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin (Sigma, US).
pCAGGSP7/NA was constructed by cloning the NA gene from the influenza virus strain A/Shanghai/2/2013 (H7N9) into expression vector pCAGGSP7, as described in our previous studies [15–20 (link)]. The plasmid was propagated in Escherichia coli XL1-blue bacteria and purified using Qiagen Purification Kits (Qiagen, US).
8
Production of Anti-Asaia Monoclonal Antibodies
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The monoclonal anti-Asaia IgG (anti-Asaia mAbs) was prepared following Kohler and Milstein methods [26 (link)]. Briefly, Balb/c mice were immunized using viable Asaia SF2.1 cells. The hybridoma was generated by the fusion of cells from murine myeloma cell line sp2/0-Ag-14 (ATCC, Rockville, MD, USA) with spleen cells from an immunized mouse. Handling of animals was performed under the control of the Ethical Committee of the University of Camerino, protocol number 2/2014, according to the Italian and European rules. Anti-Asaia antibody specificity was verified via enzyme-linked immunosorbent assay (ELISA) and immunostaining assays against various targets, including Asaia (SF2.1 strain), Gluconobacter oxydans, Acetobacter aceti (strains belonging to the Acetobacteraceae family) and Escherichia coli as an outgroup.
9
Hybridoma of Anti-Amyloid-Beta Antibody
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The hybridoma of anti-Aβ1–42 mAb clone 3F5 was generated through intraperitoneal immunization of 6–7 week old Balb/C mice with an emulsion of full length human Aβ1–42(Sequece: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) and Complete Freund’s Adjuvant (Sigma, F5881). The immunization was boosted three times intraperitoneally with full length human Aβ1–42 in incomplete Freund’ Adjuvant (Sigma, F5506). The spleen of the immunized mouse was isolated and the cells were dispersed with a 200mu mesh under sterile condition. The spleen cells were mixed with the myeloma cell line Sp2/0-Ag14 (ATCC, CRL-1581) at a 10:1 ratio, while fusion reagent Polyethylene Glycol 1500 solution (Roch 783641) was added dropwise into the mixture. After plating the cell mixture to microwell plates, the hybridomas were selected by adding HAT Supplement (Gibco, 31062–011) to the medium. Anti-Aβ1–42 antibody secreting clones were screened and subcloned based on the reaction in full length human Aβ1–42 coated ELISA plates. The isotype of the clone 3F5, as determined using the Mouse Typer Isotyping Kit (Bio-Rad, 17–2055), is IgG2b Kappa. The antibody was purified using Gammabind Plus Sepharose (GE Healthcare, 17-0886-02). ELISA plates coated with different human Aβ peptides were used to analyze the reactivity of mAb clone 3F5.
10
Mouse Myeloma Cell Line Production
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A six-week-old BALB/c female mouse was provided by the Charles River Laboratories (supplier in Poland—AnimaLAB, Poznań, Poland). The mouse was housed under controlled conditions and provided with food and water ad libitum. According to Polish law, all animal procedures were performed specifically to the Act on the Protection of Animals used for Scientific or Educational Purposes (D20150266L), which implements the European Parliament’s Directive and the Council (2010/63/EU). All procedures agreed with the Institutional Animal Care and Use Committee (IACUC) guidelines and were approved by the 2nd Local IACUC in Kraków.
Mouse myeloma cell line SP2/0-Ag14 (ATCC CRL-1581) was purchased in American Type Culture Collections (Manassas, VA, USA). Mouse hybridoma cell line B2G8 producing monoclonal antibody specific to CEA was obtained in the Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University (Krakow, Poland).
Mouse myeloma cell line SP2/0-Ag14 (ATCC CRL-1581) was purchased in American Type Culture Collections (Manassas, VA, USA). Mouse hybridoma cell line B2G8 producing monoclonal antibody specific to CEA was obtained in the Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University (Krakow, Poland).
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