For plasmid transfection, 1.5 × 105 HEK293T/17 cells or 0.8 × 105 HAP1 cells were seeded onto a TC-treated 24-well plate 1 day before transfection, and transfection was conducted when cell confluency reached 60%–70%. A total of 2 μg plasmids (1.5 μg BEs with 500 ng gRNAs or 1.5 μg PEs with 500 ng pegRNAs) were delivered using 3 μL Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. For the PE3 and PE3b experiments, 166 ng plasmids encoding gRNAs were additionally delivered to the cells. To correct the HPRT1 c.333_334ins(A) mutations in LNS patient-derived fibroblasts, 1 × 105 patient-derived fibroblasts were pulsed twice with 20 ms width and 1,200 voltage using Neon Transfection System 10 μL Kit (Thermo Fisher Scientific). For the functional assessment of HPRT1, cells were selected for 10 days in 10 μg/mL 6-TG (Sigma-Aldrich) and a medium supplemented with HAT Media Supplement (50×) Hybri-Max (Sigma-Aldrich).
Hat media supplement 50 hybri max
Manufactured by Merck Group
Sourced in United States
HAT Media Supplement (50×) Hybri-Max is a laboratory reagent used in cell culture applications. It is a concentrated solution that provides essential components required for the growth and maintenance of hybridoma cells.
Automatically generated - may contain errors
2 protocols using hat media supplement 50 hybri max
1
Plasmid Transfection and HPRT1 Correction
2
Quantifying HPRT-Mutant Cells in CHO Cultures
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CHO cells were cultured in DMEM containing HAT (hypoxanthine–aminopterin–thymidine; HAT Media Supplement (50×) Hybri-Max™; Sigma-Aldrich, St. Louis, MO, USA) for a week to eliminate preexisting HPRT-mutant cells from the culture. CHO cells were treated with the previously specified inhibitor molecules and exposed to 0–25 mJ/cm2 UVB. Cells were cultured for one more week and then harvested with trypsin-EDTA (Biosera, Budapest, Hungary). In the case of each sample, an equal number of cells (1 × 106) were seeded into 100 mm Petri dishes in selective DMEM containing 5 μM 6-thioguanine (6-TG; Sigma-Aldrich, St. Louis, MO, USA). The 6-TG-resistant cells were allowed to form visible clones for 10 days. Clones were washed with PBS, fixed with 100% methanol (Sigma-Aldrich, St. Louis, MO, USA) for 10 min, and stained with May–Grünwald–Giemsa (Molar Chemicals, Halásztelek, HU, Hungary). HPRT-mutant colonies were counted. For the positive control, 10 μM 1-methyl-3-nitro-1-nitrosoguanidine (MNNG; TCI Europe N.V., Zwijndrecht, Belgium) was used.
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