BALB/c mice (license number: SCXK2015-0018) were purchased from Hubei Provincial Laboratory Animal Research Centre (Wuhan, China). Dixiezhu and toltrazuril standards were provided by Dr. Ehrenstorfer GmbH (Augsburg, Germany). Complete and incomplete Freund’s adjuvant, ELISA coating buffer (20×), phosphate-buffered saline (PBS; 10 ×), Tris-buffered saline containing 0.1% (v/v) Tween-20 washing solution (10×), blocking solution with bovine serum albumin (BSA), an EL-Tetramethylbenzidine Chromogenic Reagent Kit, ELISA stop buffer, and enzyme-labeled secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG) were all obtained from Shanghai Shenggong (Shanghai, China). Hypoxanthine-thymidine and hypoxanthine-aminopterin-thymidine (HAT) media were purchased from Sigma (St. Louis, MO, United States). A Mouse monoclonal antibody (mAb) isotyping kit was purchased from Sino Biological (Beijing, China), an RNAeasy Mini Kit (74106) was obtained from Qiagen (Dusseldorf, Germany), and the SuperScript® III First-Strand Synthesis System for RT-PCR (18080-051) was purchased from Invitrogen (Carlsbad, CA, United States). A Gel DNA Purification Kit (GK2042) was purchased from Generay (Shanghai, China) and a Plasmid miniprep kit (PD1211-02) was purchased from Biomega (Salt Lake City, Utah, United States).
Hypoxanthine thymidine
Manufactured by Merck Group
Sourced in United States
Hypoxanthine-thymidine is a cell culture supplement used to support the growth and maintenance of cell lines. It provides the necessary nutrients and metabolites for cell proliferation and survival. The core function of this product is to facilitate cell culture procedures by ensuring optimal growth conditions for a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Hypoxanthine aminopterin thymidine, by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
1 3 dimethylaminopropyl 3 ethylcarbodiimide hydrochloride, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Graphitized carbon black (gcb), by Merck Group (1 mentions)
Chloroauric acid haucl4, by Sinopharm (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
N hydroxysuccinimide, by Merck Group (1 mentions)
Sodium carbonate, by Sinopharm (1 mentions)
Sba clonotyping system hrp, by Southern Biotech (1 mentions)
Balb c mice, by Spf Biotechnology (1 mentions)
Peg 3000, by Merck Group (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
7 protocols using hypoxanthine thymidine
1
Immunogenicity Evaluation of Coccidiostat Drugs
2
Monoclonal and Polyclonal Antibody Production Against Shigella
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mAbs and pAbs against Shigella were produced and characterized as previously described ( 10 (link)
, 14 (link)
). After a booster immunization, spleen cells were collected from immunized BALB/c mice, and then fused with SP2/0 cells. The fused cells were maintained in Roswell Park Memorial Institute (RPMI 1640) medium containing 20% fetal bovine serum (FBS) and 1% hypoxanthine-aminopterin-thymidine (Sigma-Aldrich) ( 15 (link)
, 16 (link)
). Five days later, half of the medium was replaced with RPMI 1640 containing 20% FBS and 1% hypoxanthine-thymidine (Sigma-Aldrich). The hybridoma supernatants were examined via indirect enzyme-linked immunosorbent assay (ELISA) to detect Shigella-specific antibodies ( 14 (link)
, 17 (link)
).
, 14 (link)
). After a booster immunization, spleen cells were collected from immunized BALB/c mice, and then fused with SP2/0 cells. The fused cells were maintained in Roswell Park Memorial Institute (RPMI 1640) medium containing 20% fetal bovine serum (FBS) and 1% hypoxanthine-aminopterin-thymidine (Sigma-Aldrich) ( 15 (link)
, 16 (link)
). Five days later, half of the medium was replaced with RPMI 1640 containing 20% FBS and 1% hypoxanthine-thymidine (Sigma-Aldrich). The hybridoma supernatants were examined via indirect enzyme-linked immunosorbent assay (ELISA) to detect Shigella-specific antibodies ( 14 (link)
, 17 (link)
).
3
Rapid Quantification of Phytochemicals
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PBZ, CAR, ovalbumin (OVA), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), Tween-20, Freund’s adjuvants (complete and incomplete), culture media RPMI-1640, hypoxanthine aminopterin thymidine (HAT), hypoxanthine thymidine (HT), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 3,3,5,5-tetramethylbenzidine (TMB), polyethylene glycol 20,000 (PEG 20,000, 50%), goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP), N-propylethylenediamine (PSA), and graphitized carbon black (GCB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium carbonate, N-N dimethylformamide (DMF), ethyl acetate, chloroauric acid (HAuCl4), and 3-hydroxytyramine hydrochloride (DA·HCl) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The sample pad, absorbent paper, and polyvinylchloride (PVC) backing pads were purchased from Shanghai Kinbio Tech. Co., Ltd. (Shanghai, China). The nitrocellulose (NC) membrane was purchased from Sartorius Steadi Trading Co., Ltd. (Shanghai, China). Orange, grape, and cabbage mustard samples were purchased from the Tian Hong supermarket, (Nanchang, China). The 96-well polystyrene microplates and 6-well microplates were purchased from NEST Biotechnology Co., Ltd. (Wuhan, China).
4
Quantifying Mutation Frequency via HPRT Assay
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Mutation frequency was determined using hypoxanthine phosphorybosyl transferase (HPRT) assay (Johnson, 2012 (link)). Cells were grown in DMEM medium containing hypoxanthine–aminopterin–thymidine (HAT; Sigma-Aldrich) medium for 5 d to eliminate preexisting HPRT mutants. After HAT treatment, the cells were grown in DMEM containing hypoxanthine–thymidine (Sigma-Aldrich) supplement so that both the de novo nucleotide biosynthesis pathway and the salvage pathway were able to function. Cells were harvested and 5 × 105 cells were plated on 10 cm dishes containing DMEM. After 2 d, cells were irradiated with UV (50 or 75 μJ/cm2) and grown for 2 d. Cells were harvested and 106 cells were plated on 10 cm dishes containing DMEM and 6-thioguanine (6-TG) to select HPRT mutants; HPRT+ cells incorporate 6-TG into the DNA and die, and HPRT− mutants do not incorporate 6-TG into their DNA and survive. Cells were grown for 4–6 wk with fresh DMEM + 6-TG replacing old medium every 3–4 d until visible colonies formed on the plates. Final plating for colony counting was carried out in two batches of three plates each.
5
Generation of Anti-EGFRvIII Monoclonal Antibodies
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A series of anti-EGFRvIII mAbs were prepared by AbMax Biotechnology Co., Ltd, (Beijing, China). Briefly, three BALB/c mice were immunized subcutaneously three times with the above three peptides at 2-week intervals. The titer of antibodies against EGFRvIII in blood samples was determined by ELISAs using the three peptide-BSA conjugates or F98npEGFRvIII cells as the coating antigens. Next, mouse spleen cells were collected and fused with SP2/0 myeloma cells. The fused cells were cultured in 10% FBS/DMEM containing hypoxanthine-aminopterin-thymidine (Sigma, St. Louis, MO, USA). Hybridoma colonies were maintained in 10% FBS/DMEM containing hypoxanthine-thymidine (Sigma). Hybridoma supernatants were repeatedly screened by ELISA. Finally, ascites were prepared by intraperitoneal injection of mice with 2 × 106 hybridoma cells. Ascites titers were also determined by ELISA. Furthermore, analysis of the mAb subtype was performed with standard procedures provided by the protocol of the SBA Clonotyping™ System/HRP (Southern Biotechnology Associates, Inc., Birmingham, UK). The mAb with highest titer was named 4G1.
6
Immunization Protocol for BALB/c Mice
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BALB/c mice (license number: SCXK2015-0034) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). DNC and HDP standards were provided by Dr. Ehrenstorfer GmbH (Augsburg, Germany). Complete/incomplete Freund’s adjuvant, ELISA-coating buffer (20×), phosphate-buffered saline (PBS) (10×), tris-buffered saline tween washing solution (10×), blocking solution [containing bovine serum albumin (BSA)], ELISA-3,3',5,5'-tetramethylbenzidine (EL-TMB) colorimetric kits, ELISA stop buffer, and goat anti-mouse immunoglobulin G (IgG) were obtained from Shanghai Shenggong (Shanghai, China). Hypoxanthine-thymidine and hypoxanthine-aminopterin-thymidine (HAT) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Immunization Protocol Using Freund's Adjuvant
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Freund’s complete adjuvant, Freund’s incomplete adjuvant, hypoxanthine/aminopteri n/thymidine, hypoxanthine/thymidine, and PEG3000 were purchased from Sigma-Aldrich (Shanghai, China). RPMI 1640 and fetal bovine serum were purchased from Thermo Fisher Scientific (Shanghai, China). BABL/c mice were obtained from Vital Rivea Experimental Animal Technology (Beijing, China). The animal experiments were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute, and the methods were conducted in accordance with the approved animal ethics guidelines. The Animal Ethics Committee approval number was 211123-03.
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