Hypoxanthine aminopterin thymidine hat

Manufactured by Thermo Fisher Scientific

Sourced in United States

Hypoxanthine aminopterin thymidine (HAT) is a selective medium supplement used in cell culture applications. It contains hypoxanthine, aminopterin, and thymidine, which together inhibit the de novo synthesis of nucleic acids, forcing cells to rely on the salvage pathway. This results in the selective growth of only those cells that have the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which is necessary for the salvage pathway.

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Lab products found in correlation

Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions) Polyethylene glycol, by Merck Group (1 mentions) Pvdf membrane, by Beyotime (1 mentions) Anti egfr antibody, by Abcam (1 mentions) Anti akt antibody, by Abcam (1 mentions) Bovine serum albumin (bsa), by Beyotime (1 mentions) Anti erk1 2 antibody, by Abcam (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Anti stat3 antibody, by Abcam (1 mentions) Low fluorescence pvdf membrane, by Bio-Rad (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Cell culture plate, by Corning (1 mentions)

4 protocols using hypoxanthine aminopterin thymidine hat

Hybridoma Cell Generation for Monoclonal Antibody Production

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Cell fusion and mAb production were completed by modifying the Köhler and Milstein method [16] (link). The myeloma cell's immortality and the B cell's ability to produce antibodies has been combined into a single hybridoma cell as a result of the fusion study. Lymphocytes from the spleen and lymph nodes (bronchial, axillary, inguinal, popliteal, and intraperitoneal) were fused with F0 (ATCC CRL 1646) mouse myeloma cells.
Fifty percent polyethylene glycol 4000 (PEG, Roskilde, Denmark) was used as the coupling agent and after fusion, the cell mixture was resuspended in DMEM containing hypoxanthine aminopterin thymidine (HAT; Gibco, Waltham, MA), 20% fetal calf serum and antibiotics. Plated in 96-well plates and incubated overnight at 37 °C, 5% CO 2 , and 95% humidity.
Ten to fifteen days after fusion, the wells were screened for antibody selection determined by indirect ELISA. Positive wells were cloned using the limiting dilution method, and macrophages were used as feeder cells.

Kılıç G., Demirkan E, & Yücel F. (2024). Development of Anti-idiotypic Monoclonal Antibody Mimicking SARS-CoV-2 Receptor Binding Domain. Molecular biotechnology.

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Fibroblast Line GM8148 Culture

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The fibroblast line GM8148 that contains a normal HSA17, del(17), and mar(17) (Coriell Institute for Medical Research) was grown in Minimal Essential Medium (MEM) alpha, supplemented with 10% fetal bovine serum (FBS; Cellgro) and 1X antibiotic-antimycotic solution (Gibco). Somatic cell hybrids containing the normal HSA17 (L65-14A) or del(17) (L65-13A) derived from a fibroblast cell line GM8148 as previously described (Wevrick et al., 1990 (link)) were grown in MEM alpha supplemented with 10% FBS, 1X antibiotic-antimycotic, and 1X hypoxanthine-aminopterin-thymidine (HAT; Gibco). All cells were cultured at 37°C in 5% CO₂.

Sullivan L.L., Maloney K.A., Towers A.J., Gregory S.G, & Sullivan B.A. (2016). Human centromere repositioning within euchromatin after partial chromosome deletion. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology, 24(4), 451-466.

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Monoclonal Antibody Generation against rPfeno

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For the generation of mAbs directed against various epitopes of rPfeno, 6 weeks old, female BALB/c mice were injected intraperitoneally with 50 µg of purified rPfeno emulsified with Freund’s complete adjuvant. This was followed by booster injections at an interval of 3 weeks for the next 2 months. The best responder mouse was immunized with 250 μg of immunogen (rPfeno) in phosphate buffer saline (PBS) (10 mM Na-Phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4). Five days later, the splenocytes from this mouse were fused with mouse myeloma SP2/O-Ag14 cells (Sigma-Aldrich) using polyethylene glycol (Merck) as fusogen. After selection in medium containing Hypoxanthine-Aminopterin-Thymidine (HAT, Invitrogen) for a week, the resulting hybrid clones were screened for antibody secretion wherein binding of hybridoma cell culture supernatants to rPfeno was tested by ELISA. Of the fifty seven hybrid clones that showed reactivity to rPfeno in ELISA, thirty four were re-cloned by limiting dilution to obtain pure clones. Generation of hybridomas and production of hybridoma supernatants were carried out by Bioklone Biotech Pvt. Ltd, Chennai, India.

Dutta S., Tewari A., Balaji C., Verma R., Moitra A., Yadav M., Agrawal P., Sahal D, & Jarori G.K. (2018). Strain-transcending neutralization of malaria parasite by antibodies against Plasmodium falciparum enolase. Malaria Journal, 17, 304.

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Evaluating EGFR, STAT3, AKT, and ERK Signaling

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Anti-EGFR antibody, anti-STAT3 antibody, anti-AKT antibody, and anti-ERK1/2 antibody were purchased from Abcam. Fetal calf serum (FCS) was obtained from Gibco (USA). Phospho-STAT3, phospho-AKT, and phospho-ERK1/2 antibodies were purchased from CST company (USA). Bovine Serum Albumin (BSA) and PVDF membranes were purchased from Beyotime Biotechnology (Shanghai, China). Cell culture plates were purchased from Corning (New York, USA). DMEM, hypoxanthine-aminopterin-thymidine (HAT), and HT were purchased from Invitrogen (California, USA). The low-fluorescence PVDF membrane was purchased from Bio-Rad Laboratories. Unless otherwise specified, reagents were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA). Animal Experiments were performed under a project license (No.: 20200506) granted by animal ethics committee of First Hospital of Shanxi Medical University, in compliance with national guidelines for the care and use of animals.

Ren K., Wang B, & Qi Q. (2021). Development of a new EGFR antibody antagonist which exhibits potential biological effects against laryngeal cancer. Annals of Translational Medicine, 9(12), 964.

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