The nystatin protection assay was performed as previously described (Wasylnka and Moore, 2002 (link)). Briefly, confluent epithelial monolayers were challenged with 106 spores of A. fumigatus either expressing GFP or RFP cytosolic proteins for 4 h. Monolayers were then washed twice with pre-warmed PBS and incubated with 50 μg/ml of nystatin in cell culture media (DMEM or MEM for A549 or 16HBE, respectively), at 37°C, 5% CO2 for 3 h. Samples were washed twice with pre-warmed PBS and further incubated for 1 h in supplemented DMEM/MEM until analysis. Susceptibility of the strains to nystatin was verified as previously reported (Bertuzzi et al., 2014 (link)). Live-cell imaging was performed using phenol red-free DMEM/F12 Medium (Thermo Fisher Scientific) supplemented with 10% FBS, 15 mM HEPES (pH 7).
Phenol red free dmem f12 medium
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Sourced in United States
Phenol red-free DMEM/F12 medium is a cell culture medium formulation that does not contain the pH indicator phenol red. It is designed for use in various cell culture applications where the presence of phenol red may interfere with experimental conditions or analyses.
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Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Il 1β, by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Lsm 710, by Zeiss (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Human serum, by Merck Group (2 mentions)
Endotoxin free bsa, by Merck Group (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Flou 4 am, by Thermo Fisher Scientific (1 mentions)
Dextran coated charcoal, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Kgn cell line, by RIKEN BioResource Center (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Charcoal stripped fbs, by Thermo Fisher Scientific (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
23 protocols using phenol red free dmem f12 medium
1
Nystatin Protection Assay for A. fumigatus
2
Two-photon Imaging of Calcium Dynamics
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The F-127 solution and Flou4-AM (Thermo
Fisher Scientific, F14201) were mixed in equal volumes to prepare
a working stock solution. The stock solution was diluted to a working
concentration with phenol red-free DMEM/F12 medium (Thermo Fisher
Scientific, 11039021). The diluted working solution was then added
to cell samples, and samples were incubated in dark for 10–15
min. After being washed three times with PBS, the samples were placed
in phenol red-free DMEM/F12 medium for further imaging. The processed
samples were observed with the 20× or 40× water immersion
objective of a two-photon microscope (Zeiss, LSM-710). Images were
taken every 600 ms in the time series mode, and a total of 500 images
were continuously collected in one field of view. The resolution was
selected as 512 × 512. The ROI of each cell and the change of
background fluorescence intensity over time were directly extracted
with ImageJ software. The standard fluorescence intensity (ΔF) is the ROI fluorescence intensity divided by the background
fluorescence intensity.
Fisher Scientific, F14201) were mixed in equal volumes to prepare
a working stock solution. The stock solution was diluted to a working
concentration with phenol red-free DMEM/F12 medium (Thermo Fisher
Scientific, 11039021). The diluted working solution was then added
to cell samples, and samples were incubated in dark for 10–15
min. After being washed three times with PBS, the samples were placed
in phenol red-free DMEM/F12 medium for further imaging. The processed
samples were observed with the 20× or 40× water immersion
objective of a two-photon microscope (Zeiss, LSM-710). Images were
taken every 600 ms in the time series mode, and a total of 500 images
were continuously collected in one field of view. The resolution was
selected as 512 × 512. The ROI of each cell and the change of
background fluorescence intensity over time were directly extracted
with ImageJ software. The standard fluorescence intensity (ΔF) is the ROI fluorescence intensity divided by the background
fluorescence intensity.
3
Purification and Decidualization of Human ESCs
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To purify human ESCs from endometrial tissues, a standard enzyme digestion method was used as described previously [15 (link)]. Purified ESCs were cultured in phenol red-free DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 2 mmol/L Glutamax (Thermo Fisher Scientific), 10% dextran-coated charcoal-stripped fetal calf serum (Thermo Fisher Scientific), 100 μg/mL streptomycin, and 100 IU/mL penicillin at 37 °C under a humidified atmosphere of 5% CO2 in air. phenol red-free DMEM/F12 medium and dextran-coated charcoal-stripped fetal calf serum were used to remove the effects of endogenous steroid hormones. The culture media were exchanged every three days. ESCs were used for the experiments when they were nearly confluent. Immunohistochemical analysis for vimentin was performed to confirm ESC purity, and >99% vimentin-positive cells were detected in confluent ESCs as described previously [15 (link)]. For experiments, ESCs were seeded in a 6-well plate and allowed to reach confluence before treatment. Confluent cultures of ESCs were treated with E2 (10−8 mol/L) and medroxyprogesterone acetate (MPA) (10−7 mol/L) (E2 + MPA treatment) for up to 12 days to induce decidualization, as per a previously established method [14 (link),16 (link)]. Untreated cells were used as controls.
4
Preparation and Characterization of Oligomeric Aβ42
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Oligomeric Aβ42 was prepared as described before (71 (link)). Briefly, Aβ42 peptide powders were dissolved in hexafluoroisopropanol (HFIP) (Sigma-Aldrich, 52517) to remove preexisting aggregates. HFIP was evaporated in a fume hood overnight, followed by drying down in SpeedVac for 1 hour. The resulting peptide films were dissolved in dimethyl sulfoxide to prepare 5 mM stocks, sonicated in a bath sonicator for 10 min, and diluted to 100 μM in phenol-red free DMEM/F12 medium (Thermo Fisher Scientific, 21-041-025). To prepare Aβ oligomers, the stocks were incubated at 4°C for 24 hours. BV2 cells were seeded in a 24-well plate and transfected with Abi3-targeting or negative control siRNAs. Twenty-four hours after transfection, cells were treated with 200 nM oligomeric Aβ42 for 1 hour. The proteins were extracted using RIPA buffer, including protease and phosphatase inhibitors, and sonicated in a bath sonicator. The homogenates were centrifuged at 17,000g for 15 min and the supernatants were collected for Aβ measurement. Total protein amount was measured by bicinchoninic acid assay and intracellular Aβ levels were normalized by total protein amount for each sample.
5
Breast Cancer Cell Culture and Treatment
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Hormone-dependent T47-D and BT-474 human breast cancer cells were obtained from American Type Culture Collection (Manassas, VA, USA). Both cell lines are of the luminal subtype and express ER and PR, though they differ in their Her2/neu status. BT-474 cells overexpress Her2/neu. Cells were maintained and grown in a humidified atmosphere of 5% CO2 at 37°C in phenol red-free DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO, USA). Cells were harvested using 0.05% trypsin-EDTA (Thermo Fisher Scientific). For all experiments involving MPA, cells were initially treated for 24 hours with DMEM/F12 supplemented with 5% dextran-coated charcoal (DCC; Sigma-Aldrich)-treated FBS. Subsequently, cells were washed with phosphate-buffered saline (PBS) and further incubated in fresh 5% DCC-treated FBS–DMEM/F12 for the different experimental treatments. MPA (Sigma-Aldrich), RO (Tocris, Bristol, UK), and MG-132 (EMD Millipore, Bil-lerica, MA, USA) were diluted in ethanol, PBS, and dimethyl sulfoxide, respectively. Control cells were treated with an equal volume of vehicle.
6
Characterization of Human Granulosa Tumor Cell Line
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As a steroidogenic human granulosa‐like tumor cell line, the KGN cell line obtained from RIKEN BioResource Center, Ibaraki, Japan51 was used for in vitro functional study. The KGN cells were cultured in DMEM/F12 medium (Thermo Fisher Scientific) containing 10% FBS (Biological Industries, Beit Haemek, Israel) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Mice primary GCs were cultured in DMEM/F12 with 5% FBS. All cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C.
Cells were treated in the absence or presence of recombinant human or murine IFN‐γ (PeproTech, Rocky Hill, NJ, USA), TNF‐α (PeproTech, Rocky Hill, NJ, USA), or a combination for 48 h. Then the cells were evaluated in vitro for cell proliferation and apoptosis assay. For steroid hormone measurements, cells were cultured in phenol‐red free DMEM/F12 medium (Thermo Fisher Scientific) containing 10% charcoal‐stripped FBS (Thermo Fisher Scientific). After 48 hours’ culture, the culture medium was supplemented with 10 nmol/ml testosterone as a substrate for estradiol generation for 24 h. The supernatant media was retained for estradiol measurement, and cells lysates were stored at −80°C until total RNA and protein extraction.
Cells were treated in the absence or presence of recombinant human or murine IFN‐γ (PeproTech, Rocky Hill, NJ, USA), TNF‐α (PeproTech, Rocky Hill, NJ, USA), or a combination for 48 h. Then the cells were evaluated in vitro for cell proliferation and apoptosis assay. For steroid hormone measurements, cells were cultured in phenol‐red free DMEM/F12 medium (Thermo Fisher Scientific) containing 10% charcoal‐stripped FBS (Thermo Fisher Scientific). After 48 hours’ culture, the culture medium was supplemented with 10 nmol/ml testosterone as a substrate for estradiol generation for 24 h. The supernatant media was retained for estradiol measurement, and cells lysates were stored at −80°C until total RNA and protein extraction.
7
Culturing Metastatic Breast Cancer Cells
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The MDA-MB-231 (4175) LM2 breast cancer cell line is a metastatic subpopulation isolated from TNBC MDA-MB-231 cells8 (link) and maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich Co.) at 37°C/5% CO2. TNBC MDA-MB-435 cells were obtained from ATCC (Manassas, VA, USA) and maintained at 37°C in phenol red-free DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS.26 (link) Cells were harvested with 0.05% trypsin–ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific). For all in vitro experiments, cells were allowed to reach 50–60% confluence, washed in phosphate-buffered saline (PBS), and maintained in DMEM/F12 or DMEM supplemented with 10% FBS for 24 hours prior to treatment. Incubations were carried out in the presence of LU at the various concentrations described later in a medium containing 5% FBS. Control represents cells treated with vehicle alone.
8
Culture and Passage of Breast Cancer Cell Lines
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The human breast cancer cell lines MCF-7 and MCF-7/TAMR-1 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and Merck (Darmstadt, Germany), respectively. MCF-7 cells were routinely cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Burlington, ON, Canada) supplemented with 10% fetal bovine serum (FBS, Gibco) while MCF-7/TAMR-1 cells were routinely cultured in phenol red-free DMEM/F12 medium (Gibco) supplemented with 1% FBS, 6 ng/mL insulin (Gibco) and 1 µM tamoxifen (Nacalai Tesque) following the manufacturer’s recommendation. The cell cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2 to reach 70–80% confluence. The monolayer cells were detached with trypsin–ethylene diamine tetra acetic acid (trypsin-EDTA, Gibco) to prepare single cell suspensions.
9
High-Resolution Multiscale Imaging of Cells
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High-resolution, multiscale imaging was performed on an inverse microscope equipped with a Yokogawa CSUX1A1 Nipkow spinning disc unit using a PlanApochromat 63 × /1.4 NA oil-immersion objective. Images were acquired for 30 min repeating the 33 × 2 sinterval cycles (as described in Figure 1 ) with a pixel resolution of 0.211 μm. The culture medium was replaced with phenol-red free DMEM/F12 medium (Gibco) 1 h prior to imaging, and environmental conditions of 37°C and 5% CO2 were maintained throughout the process.
10
Culturing Human Granular Carcinoma Cells
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KGN cells (a human granular carcinoma cell line) were kindly provided by the Center for Reproductive Medicine, Shandong Provincial Hospital. They were cultured in Phenol Red-free DMEM/F-12 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS) (Gibco) and incubated at 37 °C in a humidified atmosphere, with 5% CO2. KGN cells were routinely subcultured every 2 or 3 days.
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