HEK293T and Saos-2 cells were grown in DMEM (Thermo Fisher Scientific) supplemented with FBS (10% v/v). Twenty-four hours prior to transfection, cells were plated at 0.3 x 105 cells/well in 96-well plates. Cells were transiently transfected with pRL-tK (2,5ng) and pCRE-Luc, NF-kB-Luc or pGL4.30 (NFAT-Luc, Promega) (25ng) along with 20ng of empty pcDNA3.1 vector, WT, L441R or W22X ZIP14 expression constructs using Fugene 6 (HEK293T cells) or ViaFect (Saos-2 cells) (Promega). Each transfection was carried out in triplicate and repeated independently in three separate experiments. Forty-eight hours after transfection, cells were lysed and firefly and renilla luciferase activity were measured on a Glomax Multi+ Luminometer (Turner Designs) using the dual luciferase reporter assay system (Promega). Finally, the ratio of the firefly and renilla luciferase measurement was calculated.
Glomax multi luminometer
Manufactured by Turner Designs
The Glomax Multi+ Luminometer is a versatile laboratory instrument that measures luminescence. It is designed to quantify light output from a variety of samples, including those generated in luciferase reporter assays, ATP assays, and other luminescence-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Prl tk, by Promega (1 mentions)
Fugene 6, by Promega (1 mentions)
Viafect, by Promega (1 mentions)
Pcre luc, by Promega (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 protocols using glomax multi luminometer
1
ZIP14 Modulates Cell Signaling Pathways
2
Wnt Signaling Pathway Modulation
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The human embryonic kidney (HEK) cell line 293T was grown in DMEM (Invitrogen) supplemented with FBS (10%v/v, Invitrogen). Twenty-four hours prior to transfection, cells were plated at 3 x 10 4 cells/well in 96-well plates. Cells were transfected using Fugene 6 (Roche Applied Science) according to the manufacturer's instructions. In HEK293T cells, Wnt1-V5 (1ng), mesdc-2 (2ng), pRL-TK (2.5ng) and pGL3-OT (50ng) constructs were cotransfected with WT or different mutant BAMBI constructs (32ng). When needed, empty pcDNA3.1 vector was added to ensure an equal total DNA amount for all transfection experiments. As a negative control, cell were transfected with mesdc-2, pRL-TK and pGL3-OT but no Wnt1 was added. Each transfection was carried out in triplicate and repeated independently in at least three separate experiments. Forty-eight hours after transfection, cells were lysed and firefly and renilla luciferase activity was measured on a Glomax Multi+ Luminometer (Turner Designs, Sunyvale, CA) using the dual luciferase reporter assay system (Promega Corporation) according to manufacturer's instructions.
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