Phenol red free dmem f12

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Phenol red-free DMEM/F12 is a basal medium designed for the in vitro culture of a variety of cell types. It is a modification of the DMEM/F12 formulation, with the omission of the pH indicator phenol red. This formulation is suitable for applications where the presence of phenol red may interfere with experimental procedures or analyses.

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Lab products found in correlation

Penicillin, by Thermo Fisher Scientific (8 mentions) Streptomycin, by Thermo Fisher Scientific (8 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions) Insulin, by Merck Group (4 mentions) S 2366, by Werfen (3 mentions) Human protein c, by Prolytix (3 mentions) Phenol red free dmem f12, by Prolytix (3 mentions) Mda mb 231, by American Type Culture Collection (2 mentions) Mcf 10a, by American Type Culture Collection (2 mentions) Sprectra max 340, by Molecular Devices (2 mentions) Horse serum, by American Type Culture Collection (2 mentions) Epidermal growth factor (egf), by Merck Group (2 mentions) Free dmem f12, by Thermo Fisher Scientific (2 mentions) Hygromycin b, by InvivoGen (2 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Hydrocortisone, by Merck Group (2 mentions) Cholera toxin, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions) Evos microscope, by Thermo Fisher Scientific (2 mentions)

32 protocols using phenol red free dmem f12

Chondrocyte Monolayer and Pellet Cultures

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C28/I2 cells at 80% confluence were detached from T75 flasks following trypsinisation, as described above. Chondrocyte monolayers were cultured by adding 3 × 10⁵ chondrocytes to a 6-well plate in complete media for 24 h. Pellet cultures were generated by taking 3 × 10⁵ chondrocytes in 1 mL of complete media and adding the cell suspension to a 15 mL falcon before centrifuging at 300× g for 5 min to pellet the cells, and then placing this upright for 24 h with lids untightened to allow for adequate gaseous exchange.
For all cultures, complete media was removed after 24 h and the cultures were washed with DPBS^−/−. Following this, serum-free and phenol-red-free DMEM/F-12 (Thermofisher Scientific, Cambridge, UK) with 1:100 Insulin-Transferrin-Selenium (ITS; Sigma-Aldrich, Gillingham, UK) and 1% P/S was added and cultures were incubated for a further 24 h. All cultures were stimulated as detailed below. In some experiments, the last 48 h of culturing was carried out in conditions containing serum; in these experiments, phenol-red-free DMEM/F-12 with 10% FBS, exosome-depleted (Thermofisher Scientific), and 1% P/S was used instead. All incubations of cultured cells were carried out at 37 °C with 5% CO₂.

Hussain M.T., Austin-Williams S., Wright T.D., Dhawan U.K., Pinto A.L., Cooper D, & Norling L.V. (2024). β1-Integrin-Mediated Uptake of Chondrocyte Extracellular Vesicles Regulates Chondrocyte Homeostasis. International Journal of Molecular Sciences, 25(9), 4756.

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Intracellular Hypoxia Dynamics in Pseudomonas Infection

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A549, 16HBE14o-, and CFBE41o- cells were independently seeded into a Millicell® EZ SLIDE with eight wells (Merck Millipore) and left until confluence. For this experiment, phenol red-free DMEM/F12 (ThermoFisher Scientific) was used to avoid autofluorescence of the medium when visualized under a microscope. Monolayers were infected with the different P. aeruginosa strains for 3 h at a MOI = 100. After infection, the cells were washed 3 times with warm 1X PBS and then incubated with a gentamicin solution supplemented with the hypoxia probe dye (Organogenix) for 30 min (~3 h time point postinfection) and 21 h (24 h time point postinfection) independently. This probe allows the detection of environments with low oxygen levels since its phosphorescence is quenched by oxygen; therefore, its signal increases in response to a low oxygen content (red fluorescence). The hypoxia probe was used following the manufacturer’s instructions (Organogenix).
Stained monolayers were visualized under a Nikon inverted fluorescence microscope ECLIPSE Ti-S/L100 (Nikon) coupled with a DS-Qi2 Nikon camera (Nikon) to detect changes in the intracellular oxygen content depending on the P. aeruginosa strain and/or lung cell type. Analysis of the images obtained was performed using ImageJ FIJI software.

Del Mar Cendra M, & Torrents E. (2020). Differential adaptability between reference strains and clinical isolates of Pseudomonas aeruginosa into the lung epithelium intracellular lifestyle. Virulence, 11(1), 862-876.

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Pituitary Explant Culture with MEHP/DEHP

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Embryos from CD-1 mice were taken out of the uterus on embryonic day 16.5 and tissue from each embryo was collected for SRY genotyping for sex determination. The whole pituitary glands were taken out and transferred to 96-well plate with 50μL phosphate buffered saline (PBS) +1% antibiotic/antimycotic solution (anti-anti, Thermo-Fisher). Pituitaries were washed twice with PBS +1% anti-anti and 50 μL of media (phenol red-free DMEM/F12 (Thermo-Fisher), supplemented with 10% charcoal-stripped fetal bovine serum (Sigma-Aldrich) and 1% anti-anti) with different treatments (0.1 μg/mL, 1 μg/mL, 10 μg/mL and 100 μg/mL MEHP/DEHP (Sigma) added to each well. The concentrations of MEHP/DEHP were picked based on previous papers describing mouse ovarian cultures with MEHP/DEHP [30 (link),31 (link)]. Pituitaries were cultured at 37 °C for 72 hours without changing the media. To co-treat the pituitary with MEHP and AhR antagonist- CH223191 (ApexBio), pituitaries were harvested and washed as described above and first treated with the antagonist dissolved in DMSO in the 37 °C incubator for 2 hours, then MEHP was added at the final concentration of 100 μg/mL. Some pituitaries were only treated with the antagonist to eliminate possible effects of the antagonist. The final concentration of the AhR antagonist was at 1μM as instructed by the manufacturer’s product catalog.

Ge X., Weis K., Flaws J, & Raetzman L. (2021). Prenatal exposure to the phthalate DEHP impacts reproduction-related gene expression in the pituitary. Reproductive toxicology (Elmsford, N.Y.), 108, 18-27.

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Visualizing SRRM2-mCherry Induction

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The SRRM2-mCherry-inducible cell line was plated in four-well LabTek chamber slides. Day 1: cells were treated with 2 μg/ml Doxycycline to induce SRRM2-mCherry expression. Day 2: cells were treated with siCon or siNups as above. Day 4: cells were treated with one drop NucBlue reagent (ThermoFisher) per 2 ml of imaging media and 1 μM Tubulin Tracker Green (Thermofisher) 45 min prior to imaging in HEPES buffered, Phenol Red-free DMEM/F-12 (ThermoFisher) containing 10% FBS. Cells were imaged on a Leica SP8 White Light Nikon Ti-E widefield inverted microscope at 60× objective.

Strohacker L.K., Mackay D.R., Whitney M.A., Couldwell G.C., Sundquist W.I, & Ullman K.S. (2021). Identification of abscission checkpoint bodies as structures that regulate ESCRT factors to control abscission timing. eLife, 10, e63743.

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Immortalized Human Myometrial Cell Culture

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Telomerase-immortalized human myometrial (hTERT-HM) cells (52 (link)) were cultured in phenol-red free DMEM/F12 (ThermoFisher Scientific) with 10% fetal bovine serum (FBS, v/v), 1% penicillin/streptomycin at 37°C in 5% CO₂. hTERT-HM cells with doxycycline (Dox)-inducible expression of ERα (wild-type, WT) and ERα mutants (S118A and S118E) were generated by lentivirus-mediated transduction using the pINDUCER20 vector, as described below.
For experiments, the cells were treated with vehicle (DMSO), 100 nM 17β-estradiol (E2, E; Sigma, St. Louis, MO, USA), 0.2 μg/mL recombinant human relaxin-2 in 0.1% BSA-PBS (Rln, R; Peprotech, Rocky Hill, NJ, USA) and a combination of E+R for the specified amount of time. Prior to treatment, the media was supplemented with 5% charcoal-dextran-treated calf serum (CDCS) for 3 days. The cells were treated with doxycycline for 24 hours prior to the different treatments.

Tripathy S., Nagari A., Chiu S.P., Nandu T., Camacho C.V., Mahendroo M, & Kraus W.L. (2024). Relaxin Modulates the Genomic Actions and Biological Effects of Estrogen in the Myometrium by Reducing Estrogen Receptor Alpha Phosphorylation. bioRxiv.

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Adipocyte Differentiation Protocol

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Reagents. Culture medium Dulbecco's modified Eagle's medium/Nutrient Mixture F12 D-MEM/F-12 (cat. no. 12400-024), trypsin-EDTA (cat. no. 25200-072) were purchased from Gibco (Thermo Fisher Scientific, Inc.); phenol red-free D-MEM/F-12 (cat. no. 90-090-PBR) was obtained from Corning (Mediatech, Inc.); heat-inactivated fetal bovine serum (FBS) from Natocor; 3-isobutyl-1-methylxanthine (IBMX, cat. no. I7018), bovine serum albumin (BSA, cat. no. 10735086001) and dexamethasone (cat. no. D1756) were purchased from Sigma-Aldrich/Merck KGaA and rosiglitazone (cat. no. ab120762) was from Abcam.

Gantov M., Pagnotta P., Lotufo C., Rindone G.M., Riera M.F., Calvo J.C, & Toneatto J. (2021). Beige adipocytes contribute to breast cancer progression. Oncology reports, 45(1).

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In vitro migration assay protocol

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In vitro migration assays were performed using uncoated 24-well chambers/microfilters (BD), as previously described^{4 (link)–7 (link)},⁸⁹. Briefly, after rehydration of the chambers, cells (2.5 × 10⁴ cells per chamber) in 500 µL phenol red-free DMEM/F12 without FBS (Life Technologies) were seeded onto the upper chamber. In the lower chamber, 750 µL phenol red-free DMEM/F12 plus 10% charcoal-stripped FBS (Life Technologies) were added. IL-1β and/or TNFα at the indicated concentrations or vehicle only was then added into the upper chamber. Cell motility/migration was measured as the number of cells that migrated from a defined area of the uncoated microfilter through micropores in 48 h. The micropore filters were stained with 0.5% crystal violet, and the number of cells that migrated through filters was counted in the entire area of each filter. To count cell numbers objectively, a computerized image analysis system consisting of a light microscope (Leica, Lyon, France) (X20 objective, X10 ocular) and a color charge-coupling device camera (Sony, Paris, France) were utilized. All experiments were performed in duplicate.

Matsuzaki S., Pouly J.L, & Canis M. (2020). Dose-dependent pro- or anti-fibrotic responses of endometriotic stromal cells to interleukin-1β and tumor necrosis factor α. Scientific Reports, 10, 9467.

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Breast Cancer Cell Line Cultivation and Stimulation

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Human breast cancer cell lines (MCF-7, ZR-75-1, BT474, SK-BR-3, MDA-MB-453, MDA-MB-231), a mammary epitherial cell line (MCF-10A), colorectal cancer cell lines (HCT116, SW480), and epidermoid carcinoma cell line (A431) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The KPL-3C cells were established, characterized, and kindly provided by Dr. Jun-ichi Kurebayashi (Kawasaki Medical School)38 (link). All of the cell lines were cultured under the respective depositor's recommendations. The MCF-7 cells were seeded in 48-well plates (2 × 10⁴ cells mL⁻¹), 24-well plates (1 × 10⁵ cells mL⁻¹), 6-well plates (3 × 10⁵ cells 2 mL⁻¹), or 10-cm dishes (2 × 10⁶ cells 10 mL⁻¹) in MEM (Life Technologies, Rockville, MD, USA) supplemented with 10% FBS (Nichirei Biosciences, Tokyo, Japan), 1% antibiotic/antimycotic solution (Life Technologies), 0.1 mM NEAA (Life Technologies), 1 mM sodium pyruvate (Life Technologies), and 10 μg mL⁻¹ insulin (Sigma, St. Louis, MO, USA). The cells were maintained at 37°C with 5% CO₂. The next day, the medium was changed to phenol red-free DMEM/F12 (Life Technologies), supplemented with FBS, antibiotic/antimycotic solution, NEAA, sodium pyruvate, and insulin. After 24 h, the cells were treated with 10 nM 17-oestradiol (E2, Sigma) ± xanthohumol or peptides (e.g., ERAP).

Yoshimaru T., Komatsu M., Tashiro E., Imoto M., Osada H., Miyoshi Y., Honda J., Sasa M, & Katagiri T. (2014). Xanthohumol suppresses oestrogen-signalling in breast cancer through the inhibition of BIG3-PHB2 interactions. Scientific Reports, 4, 7355.

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Assessing Scaffold Thrombogenicity via Protein C Activation

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To assess scaffold thrombogenicity or the potential of the scaffolds to clot, we examined protein C activation as a surrogate for activation of the anticoagulation pathway. We adapted a previously described endothelial cell thrombomodulin assay for our studies [26] (link), [27] . Briefly, on the last day of culture, scaffolds were washed three times by retrograde perfusion of phenol red-free DMEM/F12 (Life Technologies) at 1 mL/min for a total of 45 minutes (15 minutes per wash) through the aorta. Then, we continuously circulated 4 mL of phenol-red free DMEM/F12 containing human α-thrombin (0.1 NIH U/mL, Haematologic Technologies) and human protein C (12 µg/mL, Haematologic Technologies) retrograde through the aorta of the scaffolds for 45 min at 1 mL/min. We transferred 100 µL of the medium in triplicate to a 96-well plate; sample-containing wells were mixed with 50 µL of hirudin stock (12 ATU/mL, American Diagnostica), and then the plate was incubated for five minutes at 37°C. Next, the substrate S-2366 (Chromogenix) was added to a final concentration of 0.75 mM, and the plate was incubated at room temperature for five minutes. Finally, the absorbance at 410 nm and 490 nm was measured using a Spectra MAX 340 (Molecular Devices). The relative absorbance was calculated (A₄₉₀-A₄₁₀) and normalized to the relative absorbance measured for acellular scaffolds

Robertson M.J., Dries-Devlin J.L., Kren S.M., Burchfield J.S, & Taylor D.A. (2014). Optimizing Recellularization of Whole Decellularized Heart Extracellular Matrix. PLoS ONE, 9(2), e90406.

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Imaging of Cdc42 Activity in Migrating Cells

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FRET imaging of the Cdc42 activity in randomly migrating HT-1080 cells was performed as described previously [27 (link), 38 (link)]. Briefly, HT-1080 cells expressing FRET biosensors were suspended using trypsin, plated on collagen-coated 35-mm glass-bottomed dishes, and cultured with Phenol Red-free DMEM/F12 (Invitrogen) containing 10% FBS for approximately 1 hour. Prior to imaging, the culture medium was overlaid with mineral oil to prevent evaporation. The cells were imaged with an inverted microscope (IX81; Olympus, Tokyo, Japan) equipped with an UPLANSAPO 60x oil-immersion objective lens (Olympus), a cooled charge-coupled device (CCD) camera (Cool SNAP-K4; Roper Scientific), a light-emitting diode (LED) illumination system (CoolLED precisExcite, Molecular Devices), and an IX2-ZDC laser-based autofocusing system (Olympus) with an MDXY30100T-Meta automatically programmable XY stage (Sigma Koki, Tokyo, Japan). The following filters were used for the dual-emission imaging studies: excitation filters, 435/20 for cyan fluorescent protein (CFP) and FRET (Olympus); dichroic mirrors, XF2034 (Omega); and emission filters, 480AF30 for CFP (Omega) and 535AF26 for FRET (Omega). The exposure time was 200–400 milliseconds for CFP and the FRET images when the binning was set to 4x4.

Yamao M., Aoki K., Yukinawa N., Ishii S., Matsuda M, & Naoki H. (2016). Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules. PLoS ONE, 11(10), e0164254.

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