The mouse lung epithelial cell line MLE-15 was provided by Dr. Jining Lu in the Department of Medicine, Columbia University. The pancreatic beta cell line HIT-T15 was purchased from ATCC. Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS. The pCMV-Isl1 plasmid was purchased from OriGene. To test whether the Nkx2.1 promoter is regulated by Isl1, a 0.4 kb promoter region of the Nkx2.1 gene was cloned into the pGL3 luciferase report vector, and then co-transfected into cells with pCMV-YFP or pCMV-Isl1 plasmids. The following primers were used: forward, 5’-CGTGAAGGTACCCTCTCTTTGAGACCTAAA-3’; reverse, 5’-GCAAGTCTCGAGACATGATTCGGCGTCGGC-3’. Point mutation of the potential binding sites was generated by site-directed mutagenesis using GeneArt Site-Directed Mutagenesis Kit (Thermo Fisher Scientific) with the following primers: forward, 5’-GTCATCAGCATGTAAGCTAATTATCTCGGGCAAGATGT-3’; reverse, 5’-ACATCTTGCCCGAGATACGGCGCTTACATGCTGATGAC-3’. Luciferase activity was determined 48 hours after transfection using the Dual-Luciferase Reporter Assay Kit (Promega) and the GloMax-Multi Detection System (Promega).
Hit t15
Manufactured by American Type Culture Collection
Sourced in United States
The HIT-T15 is a laboratory equipment designed for the maintenance and storage of cell cultures. It is a temperature-controlled incubator that provides a stable environment for the cultivation of various cell types. The core function of the HIT-T15 is to maintain optimal temperature, humidity, and atmospheric conditions required for the growth and preservation of cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Geneart site directed mutagenesis kit, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
2 protocols using hit t15
1
Isl1 Regulates Nkx2.1 Promoter Activity
2
Culturing GPR119-Expressing Cell Lines
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A Chinese hamster ovarian (CHO-K1) cell line stably expressing human GPR119 was purchased from Invitrogen (Carlsbad, CA, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 0.1 mM non-essential amino acids (NEAA), 25 mM HEPES (pH 7.2), 100 U/mL penicillin, 100 μg/mL streptomycin, 100 ug/mL zeocin, and 600 ug/mL hygromycin (Invitrogen). The hamster insulinoma cell line HIT-T15 was purchased from ATCC (Manassas, VA, USA) and was cultured in Ham’s F12K medium with 10% horse serum, 2.5% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). The murine L cell line GLUTag (a gift from University, Incheon, Korea) was cultured in DMEM with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). All cell lines were incubated in 5% CO2 in a humidified atmosphere at 37°C.
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