After polarization into M0, M1, M2a and M2c MDMs, the cells were stimulated with 25-hydroxy-vitamin D. Before stimulation, the media was removed, and cells were washed carefully with 10 mL PBS. 25-hydroxy-vitamin D (Cayman Chemical. Item no: 9000683. Mw 400.6) was added in a concentration of 0, 100, 1000 or 10,000 nanomolar (nM) (see Table 1 ). All vitamin D stimulations were performed in RPMI1640 with 10% charcoal stripped FCS (Thermo Fisher). After 5 min, 1 mL of the media was removed, centrifuged and stored at −80 °C until further analysis. Cells and media were placed in an incubator (5% CO2, 37 °C) for 24 or 48 h. After incubation, the media was removed and centrifuged, and the supernatant was stored at −80 °C until further analysis.
Charcoal stripped fcs
Manufactured by Thermo Fisher Scientific
Sourced in United States
Charcoal stripped FCS is a processed form of Fetal Calf Serum (FCS) that has been treated with activated charcoal to remove small hydrophobic molecules, such as hormones, steroids, and lipids. This process produces a serum that is depleted of these components.
Automatically generated - may contain errors
Lab products found in correlation
8 bromoadenosine 3 5 cyclic monophosphate, by Merck Group (2 mentions)
Medroxyprogesterone acetate, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dfc365 fx, by Olympus (1 mentions)
Mineral oil, by Irvine Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Thincert cell culture insert, by Greiner (1 mentions)
17β estradiol e, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
5 protocols using charcoal stripped fcs
1
Vitamin D Modulation of Macrophage Polarization
2
Decidualization of human embryonic stem cells
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hESCs were cultured in C-medium at 37 °C, 5% C02, and atmospheric oxygen concentrations. After reaching confluency, hESCs were passaged to start decidualization. In single wells of 48-well plates, 30,000 non-decidualized hESCs were seeded and decidualized for 5 consecutive days in DMEM/F12 phenol red-free medium supplemented with 10% heat-inactivated charcoal-stripped FCS (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (Sigma-Aldrich, Saint Louis, MO, USA), 1 μM medroxyprogesterone acetate (Sigma-Aldrich, Saint Louis, MO, USA), and 1% penicillin/streptomycin (D-medium). After six passages, new hESCs were isolated.
3
hESC Decidualization and Migration Assay
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Thirty thousand thawed hESCs were cultured in single wells of a 48-well plate and decidualized by adding DMEM/F12 phenol red-free medium supplemented with 10% heat-inactivated charcoal stripped FCS (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (Sigma-Aldrich, Saint Louis, MO, USA), 1 µM medroxyprogesterone acetate (Sigma-Aldrich, Saint Louis, MO, USA) and 1% penicillin/streptomycin (D-medium) for 5 consecutive days. Monolayers of hESCs were scratched with a p200 pipet tip to create a migration zone. hESCs were then cultured in 90 µL of pooled ECM derived from five individual embryos. Cultures were overlaid with 150 µL mineral oil (Irvine Scientific, Santa Ana, CA, USA) to prevent evaporation. For addition of RNase, we used 1 mg/ml of RNase in PBS (10109169001, Roche, Basel, Switzerland). At 0 and 18 h after creation of the migration zone, phase contrast images were taken using a monochrome digital camera (Leica DFC365 FX) attached to an inverted microscope (Olympus IX71) with a U Plan FL 4x/0.13 PhL objective. The migration response of hESCs was quantified by assessing the reduction of the surface area of the migration zone by using Image J software (version 1.50i).
4
AR CALUX Cell Cultivation and Assay
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AR CALUX® cells were obtained and used under license from BDS. The cells were cultured in growth medium comprised of Dulbecco’s Modified Eagle Medium (DMEM/F12, Thermofisher 31331028) containing 7.5% heat inactivated fetal calf serum (FCS), 1% non-essential amino acid solution (NEAA, Sigma), and 10 000 U/ml penicillin/10 000 µg/ml streptomycin (Sigma). During subculture, once per week 200 µg/ml G418 (gentamycin, Sigma) solution was added to the medium. The assay was performed in Phenol Red-free DMEM/F12 medium (Thermofisher 21041025) containing 5% charcoal stripped FCS (Gibco), 1% NEAA, and 10 000 U/ml penicillin/10 000 µg/ml streptomycin.
5
Investigating Dienogest and Estradiol Effects
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All cell types were cultured in a humidified incubator at 37 °C and 5% CO2, and the medium was routinely renewed every 2–3 days. Cells were washed once with PBS and passaged at about 80% confluence. All cell culture reagents were purchased from Invitrogen/ThermoFisher Scientific (Karlsruhe, Germany) unless stated otherwise. Treatments with hormones were conducted with charcoal-stripped FCS (Gibco, Cat. No. 12676-029). Depending on the scope of the experiment, cells were cultured in 6-well plates or ThinCert cell culture inserts (Greiner, Kremsmünster, Austria) and serum-starved (medium + 1% FCS) for 24 h. Cells were pre-treated in duplicates with either vehicle (ethanol) or dienogest (DNG, 20–100 nM, Sigma-Aldrich) 2 h prior to stimulation with 17β-estradiol (ES, 10–50 nM, Sigma-Aldrich) for various times.
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