Shh-LIGHT2 cells were seeded in 96-well plates at a density of 3.5 × 104 cells/well. After 24 h, growth media was replaced with serial dilutions of 2 , 20(S)-OHC, 20(R)-OHC, or SAG (CAS no. 912545-86-9, Carbosynth, FS76762) in low-serum media (phenol red-free high-glucose DMEM, Gibco, 21063045) containing 0.5% bovine calf serum (CS, ATCC, CRL-1658), 100 U/mL of penicillin-streptomycin (Gibco, 15140163), and 1 mM sodium pyruvate) at a normalized DMSO concentration of 0.2%. After 30 h, cells were washed with PBS and treated with Passive Lysis Buffer (20 μL/well, Promega, E1941) at room temperature for 15 min with rocking. 10 μL lysate from each well was transferred to a white-bottomed assay plate (Corning, 3912) for Firefly and Renilla luciferase measurements using a Dual Luciferase Reporter kit (Promega, E1960) on a Tecan Spark M10 multimode plate reader. Gli activity was calculated as the ratio of Firefly/Renilla luciferase signal and percent Gli activation was assessed relative to DMSO-only control values. Dose-response curves were generated using GraphPad Prism software.
Phenol red free high glucose dmem
Manufactured by Thermo Fisher Scientific
Phenol red-free high-glucose DMEM is a cell culture medium formulation that does not contain phenol red, a pH indicator dye, and has a high concentration of glucose. This medium is commonly used to support the growth and maintenance of various cell lines in vitro.
Automatically generated - may contain errors
Lab products found in correlation
White bottomed assay plate, by Corning (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Dual luciferase reporter kit, by Promega (2 mentions)
Spark m10, by Tecan (2 mentions)
Charcoal stripped fcs, by Merck Group (1 mentions)
3 protocols using phenol red free high glucose dmem
1
Quantifying Hedgehog Signaling Modulation
2
Quantifying Hedgehog Signaling Modulation
3
Estrogen Response in MCF-7 Breast Cancer Cells
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The MCF-7 human breast cancer cell line originates from a 69-year old Caucasian woman and is estrogen receptor (ER) positive, progesterone positive (PR) and HER2 negative. Here MCF-7 cells (a clonal isolate obtained from the ATCC (catalogue number HTB-22) kindly provided by Prof. Edison Liu, Jackson Laboratories, Maine, USA) were grown in 15 cm plates to 80% confluency. Plates were then washed two times with PBS and overlaid with 20 ml of phenol-red free high glucose DMEM (Gibco) containing 2% charcoal stripped FCS (Sigma). After 24 h of incubation, the cells were again washed with PBS and fresh media containing 2% charcoal stripped FCS was added. This process was repeated over a three day period to generate cells devoid of estrogen. The time course (5, 10, 20, 40, 80, 160, 320, 640 and 1280 min) was initiated by replacing media with prewarmed media containing 10 nM E2. In addition, an untreated sample was included in the experiment as a zero time point.
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