For treatments in full serum, cells were plated in 96-well plates and treated in duplicate wells with a dose titration of CPI-1612 for 4 days. For treatments in charcoal-stripped serum, cells were plated in 96-well plates, washed after overnight incubation, and moved to phenol-red free media (Gibco) + 10% charcoal-stripped serum (Gibco) for 2 days. 17-β-estradiol at 100 nM was added along with a dose titration of CPI-1612 for 4 days. Viability was assessed using Cell Titer Glo (Promega), and GraphPad Prism curve fitting was used to fit the data.
Charcoal stripped serum
Manufactured by Thermo Fisher Scientific
Sourced in United States
Charcoal-stripped serum is a laboratory product used to remove small molecules, hormones, and other components from biological samples. It is often used in various research and analytical applications to create a more controlled environment for experimental studies.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 glutamax, by Thermo Fisher Scientific (3 mentions)
Lncap, by American Type Culture Collection (3 mentions)
17β estradiol e2, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Phenol red free media, by Thermo Fisher Scientific (2 mentions)
Celltiter glo, by Promega (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Fetal calf serum (fcs), by Greiner (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
Lncap, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
4 hydroxytamoxifen, by Merck Group (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Phenol red free rpmi 1640, by Thermo Fisher Scientific (1 mentions)
28 protocols using charcoal stripped serum
1
Dose-Response Viability Assay in Breast Cancer Cells
2
Evaluating CPI-1612 Cytotoxicity in Breast Cancer
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For treatments in full serum, cells were plated in 96-well plates and treated in duplicate wells with a dose titration of CPI-1612 for 4 days. For treatments in charcoal-stripped serum, cells were plated in 96-well plates, washed after overnight incubation, and moved to phenol-red free media (Gibco) + 10% charcoal-stripped serum (Gibco) for 2 days. 17-β-estradiol at 100 nM was added along with a dose titration of CPI-1612 for 4 days. Viability was assessed using Cell Titer Glo (Promega), and GraphPad Prism curve fitting was used to fit the data.
3
Cell Line Cultivation Conditions
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T47D-KBluc and MDA-MB-453 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The MCF7 and HeLa cell lines were a gift from Dr Sandra Dunn (Division of Hematology and Oncology, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada). TamR cell lines TamR3 and TamR6 were kindly provided by Dr Euphemia Leung (University of Auckland, New Zealand) [45 (link)]. Cells were cultured at 37°C in a humidified incubator with 5% CO2. The cell lines were maintained in the following culture media: for MCF7, phenol red–free RPMI 1640 (Gibco/Life Technologies, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS) (Gibco/Life Technologies); for T47D-KBluc, phenol red–free RPMI 1640 containing 4.5 g/L glucose (Sigma-Aldrich, St Louis, MO, USA), 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Sigma-Aldrich), pH 7.5, 1 mM sodium pyruvate (Life Technologies), 0.2 U/ml insulin (Sigma-Aldrich) and 10% FBS; for MDA-MB-453 and HeLa, Dulbecco’s modified Eagle’s medium (HyClone Laboratories/GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% FBS (Gibco/Life Technologies); and for TamR3 and TamR6, phenol red–free RPMI 1640 containing 10% charcoal-stripped serum (CSS) (Gibco/Life Technologies) and 1 μM tamoxifen (Sigma-Aldrich).
4
Corticosterone Rhythms in Brown Adipocytes
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Murine immortalized preadipocytes were generated and cultured as previously described [22 (link)]. Pre-adipocytes were differentiated for 14–15 days. During the last 2 days of differentiation and during the experiments, cells were grown in basal culture medium containing hormone-deprived (charcoal-stripped) serum (Gibco). To investigate whether corticosterone (Sigma–Aldrich) can induce rhythm in brown adipocytes, cells were pretreated with or without 1 μM of GR antagonist RU486 (Sigma–Aldrich) for 30 min, followed by treatment with 10 nM of corticosterone or vehicle, and cells were harvested at T = 0, 8, 16, 24, or 32 h after treatment for gene expression analysis.
5
Hormone-responsive Vascular Tissue Engineering
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HUVEC and HLF were expanded using the same techniques used in 2D culture. PDA treated PDMS microarrays were loaded with 2 ×106 cells/ml HLF and 2 ×106 cells/ml HUVEC in a hydrogel comprised of blended collagen type I (2.25 mg/ml collagen type I, Corning) and fibrin (5mg/mL fibrinogen activated with 1 U/ml thrombin, Sigma Aldrich). Hydrogels were allowed to solidify at 37°C for 30 minutes. Stromal vascular tissues were cultured in vascular cell growth media supplemented with VEGF (ATCC), 25 μg/mL aprotinin (Sigma Aldrich), and charcoal stripped serum (Gibco) for 48 hours. Media was changed to phenol red free media containing 2% charcoal stripped serum for complete hormone starvation for 24 hours before exposing the gels to 0nm, 1nm, or 10nm E2 in ethanol or 0nM, 1nM, or 10nM DHT for 72 hours.
6
Cell Culture and Compound Preparation Protocol
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MM1.S, MM1.R, CEM-C7-14 and CEM-C1-15 cells were cultured in RPMI1640 GlutaMAX (Gibco, life technologies), supplemented with 10% fetal calf serum (Greiner bio-one), 100U/ml penicillin and 0.1mg/ml streptomycin (Gibco, life technologies), and grown in a 5% CO2 incubator at 37°C. MM1.S, MM1.R were purchased from ATCC, CEM-C7-14 (C7-14) and CEM-C1-15 (C1-15) cells were a kind gift from Prof. Brad E. Thompson (University of Texas Medical branch). All experiments were performed using charcoal-stripped serum (Gibco, life technologies). All cell lines were regularly tested for mycoplasma contamination and were negative.
Dexamethasone (Dex) was purchased from Sigma Aldrich, dissolved in ethanol (EtOH) and stored at -20°C. Compound A (CpdA) was synthesized as described by Louw et al.[26 (link)], dissolved in EtOH, flushed with an inert gas (N2-vapours), protected from light and was stored at -80°C. Recombinant murine TNFα, obtained from the VIB protein service facility, was dissolved in cell culture medium and used at a final concentration of 2000IU/ml. The total solvent concentration in all experiments was kept equal in each condition.
Dexamethasone (Dex) was purchased from Sigma Aldrich, dissolved in ethanol (EtOH) and stored at -20°C. Compound A (CpdA) was synthesized as described by Louw et al.[26 (link)], dissolved in EtOH, flushed with an inert gas (N2-vapours), protected from light and was stored at -80°C. Recombinant murine TNFα, obtained from the VIB protein service facility, was dissolved in cell culture medium and used at a final concentration of 2000IU/ml. The total solvent concentration in all experiments was kept equal in each condition.
7
Prostate Cancer Cell Line Cultivation
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The LNCaP human prostate cancer cell line was sourced from the Chinese Academy of Sciences Shanghai Cell Repository (Shanghai, China), and the C4-2 human prostate cancer cell line was sourced from Dr Donghui Han (Department of Urologic Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, China), who obtained from the China Center for Type Culture Collection (Wuhan, China). Fetal bovine serum (FBS) and charcoal stripped serum were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Gene-specific primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 2 mmol/l glutamine, 0.06 g/l penicillin, 0.1 g/l streptomycin and 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 5% CO2.
8
Prostate Cancer Cell Line and PDX Cultivation
9
Multimodal Cellular Stress Response
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A triplex test simultaneously evaluating cellular redox potential (data not shown), proliferation (data not shown), and apoptosis of the cells was used in order to reduce population and systematic discrepancies between cell cultures. Apoptosis was assessed with the DELFIA® DNA fragmentation assay (PerkinElmer), which exploit terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL reaction) and samarium-labeled streptavidin.
Ten thousand cells/well were incubated for 48 h in culture media deprived of phenol red, supplemented with 10% charcoal stripped serum (Gibco) on a white, clear bottom 96-well plates. Cells were then treated either with 10−8 M 17β-estradiol (Sigma) or vehicle control (i.e., equivalent amounts of ethanol). Samples were collected at time 0 (untreated) and 24 h after treatment.
Ten thousand cells/well were incubated for 48 h in culture media deprived of phenol red, supplemented with 10% charcoal stripped serum (Gibco) on a white, clear bottom 96-well plates. Cells were then treated either with 10−8 M 17β-estradiol (Sigma) or vehicle control (i.e., equivalent amounts of ethanol). Samples were collected at time 0 (untreated) and 24 h after treatment.
10
Sex-specific fibroblast and endothelial response to E2 and DHT
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Primary human lung fibroblasts (HLF), human dermal fibroblasts (HDF), and human umbilical vein endothelial cells (HUVEC) from both XX and XY donors were sourced for use in these studies (ages 20–45, ATCC). HLF and HDF were expanded in fibroblast basal medium supplemented with fibroblast growth kit – low serum (ATCC) and HUVEC were grown in vascular cell basal medium supplemented with endothelial growth kit – VEGF (ATCC). At 50% confluence, cells were changed to phenol red-free media supplemented with charcoal stripped serum (Gibco) for a 24 hour serum starvation. After 48 hours of starvation, we exposed cells to 0nM, 1nM or 10nM E2 dissolved in ethanol (Thermo Fisher). E2 was replenished every 24 hours and media was changed every 48 hours. At day 1, 2, 3, 4, and 7, cells were collected via trypsinization. These trials were repeated with 0nM, 1nM, and 10nM DHT using the same time points and methodology as was used for E2 exposures. Cell pellets were stored in RNAlater (Thermo Fisher) at 4°C until RNA isolation.
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