Each of the experiments on the ESCs was performed using cells separately prepared from the endometrial tissue specimens obtained from different patients. When the culture had reached a confluence of 70%, the ESCs were treated with serum-free, phenol red-free media (Sigma-Aldrich, St. Louis, MO, USA) for 48 h before treatment. In order to evaluate the effect of HR-RBP4 (catalog no. 3378-LC, R&D Systems) and RBP4 siRNA on cellular viability, invasiveness, the expression of MMP-2/9, and proliferation, the cultures were separately treated with the vehicle (control), 25 ng/mL HR-RBP4, 30 nmol/L negative control siRNA, or 30 nmol/L RBP4 siRNA for 48 h.
Phenol red free media
Manufactured by Merck Group
Sourced in United States
Phenol red-free media is a type of laboratory cell culture media that does not contain the pH indicator dye phenol red. This type of media is used in applications where the presence of phenol red may interfere with experimental measurements or observations.
Automatically generated - may contain errors
Lab products found in correlation
Hr rbp4, by R&D Systems (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
α mem, by Merck Group (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Estradiol, by Merck Group (1 mentions)
Charcoal stripped fbs, by Thermo Fisher Scientific (1 mentions)
Celltiter glo, by Promega (1 mentions)
Tnf α, by Merck Group (1 mentions)
6 protocols using phenol red free media
1
Effects of HR-RBP4 and RBP4 siRNA on Endometrial Stromal Cells
2
Estradiol Regulates miRNA Expression
3
Doxycycline-Induced G0S2 Proliferation
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Cells were treated with 0.5 μg/mL doxycycline for 15 days and then assessed for cell proliferation by CellTiter-Glo (Promega). The time point of 15 days of G0S2 induction was chosen as pilot experiments determined that prominent proliferative effects did not begin until 12 days of G0S2 induction. Briefly, an equal number of cells were plated in 24-well plates in four biological replicates at 20,000 cells per well, and viable cell numbers were estimated for 6 consecutive days using the CellTiter-Glo assay. Estrogen-depleted assays were performed by replacing standard media with phenol red-free media (Sigma), charcoal-stripped FBS (Invitrogen), and 1% sodium pyruvate (Corning) one day after cell plating.
4
Isolation and Treatment of ESCs
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ESCs were separated and maintained in a monolayer culture as described previously.14 (link) ESCs after first passage were assayed immunocytochemically using specific cell-surface markers, and we previously showed that the purity of isolated ESCs was more than 95%.14 (link) We utilized only the cells after the first passage in all of the experiments using ESCs.
When Ishikawa cells and ESCs were grown to 70% confluence, they were treated with serum-free, phenol red-free media (Sigma-Aldrich) for 18 h before treatment. Afterwards, cell cultures were treated with either vehicle (control), TNF-α (10 ng/mL and 25 ng/mL) or IL-1β (10 ng/mL and 25 ng/mL) for 1 h and 6 h, respectively. For treatment with peritoneal fluid, 1×105 cells were seeded in six-well plates, and when 80% confluence was reached, the cells were starved in serum-free, phenol red-free media (Sigma-Aldrich) overnight. Afterwards, either vehicle or 25% peritoneal fluid was added, and RNA was extracted 1 h and 6 h after the addition of peritoneal fluid.
When Ishikawa cells and ESCs were grown to 70% confluence, they were treated with serum-free, phenol red-free media (Sigma-Aldrich) for 18 h before treatment. Afterwards, cell cultures were treated with either vehicle (control), TNF-α (10 ng/mL and 25 ng/mL) or IL-1β (10 ng/mL and 25 ng/mL) for 1 h and 6 h, respectively. For treatment with peritoneal fluid, 1×105 cells were seeded in six-well plates, and when 80% confluence was reached, the cells were starved in serum-free, phenol red-free media (Sigma-Aldrich) overnight. Afterwards, either vehicle or 25% peritoneal fluid was added, and RNA was extracted 1 h and 6 h after the addition of peritoneal fluid.
5
Investigating Cellular Responses in Ishikawa and ESCs
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Each experiment using Ishikawa cells was performed with cells prepared separately at 5 different time points for Pak4 siRNA transfection or 5 different time points for TNF-a and pyrrolidine dithiocarbamate (PDTC) treatment. Each experiment with ESCs was performed using cells prepared from endometrial tissue specimens obtained from 7 different patients for Pak4 siRNA transfection, and 5 different patients for TNF-a and PDTC treatment. Ishikawa cells and ESCs were grown to 70% confluence and treated with serum-free, phenol red-free media (Sigma-Aldrich) before chemical treatment.
6
NF-κB Activation in Endometrial Cells
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Ishikawa cells and ESCs were grown in serum-free, phenol redfree media (Sigma-Aldrich) for 18 hours. After this step, the cells were treated either with medium only (for the control and TNF-a groups) or 10 À5 M PDTC (Sigma) (for the TNF-a plus PDTC group) for 1 hour. Next, the cells were treated with medium only (control) or 10-nM TNF-a (Sigma) (for the TNF-a and TNF-a plus PDTC groups) for 30 minutes for the NF-kB p65-DNA (deoxyribonucleic acid) binding immunodetection assay, or for 24 hours for Pak4 western blot analysis. Nuclear protein extraction and the NF-kB p65-DNA binding immunodetection assay were performed as described in our previous study (18) . For experimental control for nuclear and cytosol fractions, we performed western blot analysis on lamin B protein (Santa Cruz Biotechnology). We could see clear lamin-B expression in the nuclear fraction (A: Ishikawa cell; B: endometrial stromal cell), whereas no expression was observed in the cytosol fraction (Supplemental Fig. 1, available online). As an experimental control for the NF-kB p65-DNA binding immunodetection assay, we performed western blot analyses on the NF-kB p65 subunit in the nuclear fraction as described in our previous study (18) .
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