On the day before transfection, HEK293 cells were trypsinized, counted, and plated onto 12-well plates at a density of 4×105 cells/well. The cells were transfected by adding a premixed solution containing 0.4 μg of expression vectors and 2 μl of ScreenFectA (Wako, Osaka, Japan). After 24 hours of incubation, the medium was exchanged twice with warmed Waymouth’s medium (Life Technologies, Carlsbad, CA, USA), and the cells were incubated for 30 minutes at 37°C in uptake medium containing [5,6,8,11,12,14,15-3H(N)]-PGE2 (PerkinElmer, Waltham, MA, USA) at 0.6 nM. The cells were washed four times with cold Waymouth’s medium, and lysed with 200 μl of RIPA Buffer (Thermo Fisher Scientific, Hemel Hempstead, UK) containing a protease inhibitor (Roche, Basel, Switzerland). The total protein concentration was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific). Next, 150 μl of cell lysate was mixed with 5 ml of MicroScint–20 (PerkinElmer), and scintillation counting was performed in a Tri-Carb 3100TR liquid scintillation spectrometer (PerkinElmer).
Waymouth s medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Waymouth's medium is a cell culture medium formulated for the in vitro cultivation of various mammalian cell types. It provides the necessary nutrients and growth factors to support the growth and maintenance of cells in a controlled laboratory environment. The medium's composition is designed to support the specific requirements of the targeted cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (17 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
Soybean trypsin inhibitor, by Thermo Fisher Scientific (2 mentions)
Insulin transferrin selenium ethanolamine its x, by Thermo Fisher Scientific (2 mentions)
Bovine pituitary extract, by Thermo Fisher Scientific (2 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Microscint 20, by PerkinElmer (2 mentions)
Screenfect a, by Fujifilm (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Skbr3, by American Type Culture Collection (2 mentions)
Bronchial epithelial growth kit, by American Type Culture Collection (2 mentions)
Dms153, by Thermo Fisher Scientific (2 mentions)
Dms114, by Thermo Fisher Scientific (2 mentions)
33 protocols using waymouth s medium
1
Radioactive PGE2 Uptake in HEK293 Cells
2
Acinar Cell Culture Establishment and Quantification
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Acinar cultures were established according to previous publications (Kurup and Bhonde, 2002 (link); Means et al., 2005 (link); Ardito et al., 2012 (link)). Briefly, dorsal pancreata were minced in Hank's buffered saline solution and digested sequentially in 0.02% trypsin (5 min, 37°C) and 1 mg/ml collagenase P (Roche Applied Science, Mannheim, Germany; 15 min, 37°C), following filtration to eliminate undigested material, and repeated washing to eliminate debris and dead cells, acinar cell clusters were embedded in rat tail collagen gels (Corning, Corning, NY), and cultured in Waymouth's medium (Life Technologies, Carlsbad, CA) supplemented with 1% fetal bovine serum, 0.4 mg/ml soybean trypsin inhibitor, and 1 µg/ml dexamethasone. Cultures were fixed and imaged after 5 days. To quantify cyst size, we randomly selected >10 fields per mouse, imaged, and quantified the maximal diameter of each transformed cyst using ImageJ.
3
Isolation and Culture of Primary Pancreatic Acini
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Primary pancreatic acini were isolated as described in Panciera et al. 201618 (link) from rtTAM2; colYAP mice, or from rtTAM2 littermates as control (both male and females, 6-8 weeks old). Acini were seeded in neutralized rat tail collagen type I (Cultrex)/acinar culture medium (1:1)53 (link) and overlaid with acinar culture medium (Waymouth’s medium [Life Technologies] supplemented with 0.1% FBS [Life Technologies], 0.1% BSA, 0.2 mg/ml soybean trypsin inhibitor [SBTI], 1× insulin-transferrin-selenium-ethanolamine [ITS-X] [Life Technologies], 50 μg/ml bovine pituitary extract [BPE] [Life Technologies], 1μg/ml dexamethasone [Sigma], and antibiotics) supplemented with 0.5μg/ml doxycycline and DMSO or 10μM JQ1, as indicated. ADM events were counted 2-4 days after seeding. For EdU incorporation, 20μM EdU was added to culture medium for 90 min, then collagen cushions containing acini/ducts were extensively washed in PBS and fixed in 4% PFA for 20 min at RT. EdU staining was performed with Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific), according to manufacturer instructions. Total RNA was extracted with TRIzol Reagent (Invitrogen).
4
Endothelial Cell Functional Assays
5
Isolation and Culture of Primary Pancreatic Acini
6
Western Blot Analysis of EGR-1 Knockdown
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Western blot analysis was performed with extracts of cells treated with siRNA targeting human EGR-1. SMC (80–90% confluency) were arrested in Waymouth’s medium (Invitrogen, MD) containing 0.05% FBS for 6 h. Cells were incubated with 100 nM EGR-1 siRNA, siCTL or the transfection agent DharmaFECT alone overnight. FGF2 (50 ng/ml) was added for 1 h. Total protein was harvested in radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitors44 (link). Proteins were resolved on 4–20% (w/v) sodium dodecyl sulfate (SDS)-polyacrylamide gradient gels (Bio-rad Mini-PROTEAN TGX) and transferred to Immobilon-P PVDF membranes (Millipore, USA). Membranes were blocked with 5% skim milk and incubated with primary rabbit monoclonal EGR-1 antibody (1:1000, Cell Signaling, USA) at 4 °C overnight or mouse monoclonal ß-actin antibody (1:30000, Sigma-Aldrich) at 22 °C for 1 h then incubated with a secondary goat anti-rabbit (1:1000, DAKO Cytomation, Denmark) or goat anti-mouse (1:1000, DAKO Cytomation, Denmark) antibodies for 1 h. Chemiluminescence was detected using the Western Lightning Chemiluminescence system (PerkinElmer, USA) and ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare Life Sciences, USA). Band intensity was quantitated using the Gel Analysis method in NIH ImageJ and normalized to β-actin.
7
Culturing Tumor and Macrophage Cell Lines
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Tumorigenic mouse mammary carcinoma EMT6 (ATCC CRL-2755) and 4T1 (ATCC CRL-2539) as well as J774A.1 macrophage (ATCC TIB-67) cell lines were obtained from the American Type Cell Culture Collection. EMT6 cells were maintained in Waymouths medium (Invitrogen, Carlsbad, CA, USA) containing 10% heat-inactivated fetal calf serum (FCS; Moregate, Brisbane, QLD, Australia). 4T1 and J774 cells were grown in RPMI 1640 medium (Invitrogen) + 10% FCS.
Bone marrow-derived macrophages (BMDM) were prepared by flushing femurs of 8- to 12-week-old mice with phosphate buffered saline (PBS). Bone marrow cells were seeded onto untreated culture plates and cultured for 7 days in RPMI + 10% FCS containing mouse colony stimulating factor (mCSF)-1 (50 ng/mL; BioLegend, San Diego, CA, USA). All cells were maintained at 37 °C in an atmosphere of 5% CO2 in air (Invitrogen).
Bone marrow-derived macrophages (BMDM) were prepared by flushing femurs of 8- to 12-week-old mice with phosphate buffered saline (PBS). Bone marrow cells were seeded onto untreated culture plates and cultured for 7 days in RPMI + 10% FCS containing mouse colony stimulating factor (mCSF)-1 (50 ng/mL; BioLegend, San Diego, CA, USA). All cells were maintained at 37 °C in an atmosphere of 5% CO2 in air (Invitrogen).
8
Cholesterol Depletion Impacts NetB Oligomerization
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To examine the inhibitory effect of cholesterol-interacting agents, LMH cells were
incubated at 37°C for 30 min in the presence or absence of 5 mM methyl-β-cyclodextrin
(MCD; Sigma) in 2.5 ml of Waymouth’s medium (Thermo Fisher Scientific Inc.) and were
washed twice with PBS. The removal of cholesterol was confirmed using a cholesterol
measurement kit (Cholesterol-E-Test; Wako) according to the manufacturer’s instructions.
Each cell was treated with NetB (10 µg/ml) for an additional 1 hr at 37°C. After washing,
cells were suspended in 0.1 ml of MBS containing 1% Triton X-114 and 0.1% protease
inhibitor cocktail. After incubating for 1 hr on ice, the detergent-insoluble fractions
were separated from the supernatant by centrifugation at 15,000 × g for
15 min and were subsequently resuspended in 1 ml of PBS. Samples were subject to SDS-PAGE
and immunoblotting using affinity-purified rabbit anti-NetB (10 µg/ml) or anti-actin
(Sigma) (1:100) IgG. The reactive bands were developed by chemiluminescence. The LAS-4000
system was used to visualize signals and quantified using densitometry software
(MultiGauge; FUJIFILM Corp., Tokyo, Japan). The densities of monomers and oligomers in
each lane were shown as relative values when we normalized the density of actin in the
same lane to100.
incubated at 37°C for 30 min in the presence or absence of 5 mM methyl-β-cyclodextrin
(MCD; Sigma) in 2.5 ml of Waymouth’s medium (Thermo Fisher Scientific Inc.) and were
washed twice with PBS. The removal of cholesterol was confirmed using a cholesterol
measurement kit (Cholesterol-E-Test; Wako) according to the manufacturer’s instructions.
Each cell was treated with NetB (10 µg/ml) for an additional 1 hr at 37°C. After washing,
cells were suspended in 0.1 ml of MBS containing 1% Triton X-114 and 0.1% protease
inhibitor cocktail. After incubating for 1 hr on ice, the detergent-insoluble fractions
were separated from the supernatant by centrifugation at 15,000 × g for
15 min and were subsequently resuspended in 1 ml of PBS. Samples were subject to SDS-PAGE
and immunoblotting using affinity-purified rabbit anti-NetB (10 µg/ml) or anti-actin
(Sigma) (1:100) IgG. The reactive bands were developed by chemiluminescence. The LAS-4000
system was used to visualize signals and quantified using densitometry software
(MultiGauge; FUJIFILM Corp., Tokyo, Japan). The densities of monomers and oligomers in
each lane were shown as relative values when we normalized the density of actin in the
same lane to100.
9
Isolation and Culture of Pancreatic Epithelial Cells
10
Isolation and Characterization of Membrane Rafts
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LMH cells cultured in a 25 cm2 flask were washed with PBS at 37°C and
suspended in 2 ml of NetB solution prepared at 10 µg/ml with Waymouth ’s medium (Thermo
Fisher Scientific Inc.). After incubating at 37°C for 1 hr, the mixture was washed with
PBS at 4°C and added to 0.35 ml of morpholinoethanesulfonic acid (MES)-buffered saline
(MBS; 25 mM MES, 150 mM NaCl, 2 mM EDTA, pH 6.5) containing 1% Triton X-114 and 0.1%
protease inhibitor cocktail (Sigma). After keeping the mixture on ice for 1 hr, the sample
was treated with 0.35 ml of 80% sucrose-containing MBS. Upon mixing properly, the sample
was transferred to an ultracentrifuge tube (Ultra-Clear ™ Centrifuge Tubes 11 × 60 mm;
Beckman Coulter, Brea, CA, USA), and 2 ml of 30% sucrose-containing MBS was added followed
by layered with 1.3 ml of MBS containing 5% sucrose. After ultracentrifugation using with
a SW60Ti (Beckman) at 250,000 × g for 18 hr at 4°C, 0.4 ml of fractions
were collected in order from the bottom. To concentrate the sample, 100 µl of 30%
trichloroacetic acid was added to each fraction, mixed, and allowed to stand on ice for 30
min. The mixture was centrifuged at 15,000 × g for 10 min at 4°C, and 500
µl of acetone was added to the precipitate and mixed. After centrifugation at 15,000 ×
g for 10 min at 4°C, the supernatant was removed, and the pellet was
suspended in PBS after complete evaporation of acetone.
suspended in 2 ml of NetB solution prepared at 10 µg/ml with Waymouth ’s medium (Thermo
Fisher Scientific Inc.). After incubating at 37°C for 1 hr, the mixture was washed with
PBS at 4°C and added to 0.35 ml of morpholinoethanesulfonic acid (MES)-buffered saline
(MBS; 25 mM MES, 150 mM NaCl, 2 mM EDTA, pH 6.5) containing 1% Triton X-114 and 0.1%
protease inhibitor cocktail (Sigma). After keeping the mixture on ice for 1 hr, the sample
was treated with 0.35 ml of 80% sucrose-containing MBS. Upon mixing properly, the sample
was transferred to an ultracentrifuge tube (Ultra-Clear ™ Centrifuge Tubes 11 × 60 mm;
Beckman Coulter, Brea, CA, USA), and 2 ml of 30% sucrose-containing MBS was added followed
by layered with 1.3 ml of MBS containing 5% sucrose. After ultracentrifugation using with
a SW60Ti (Beckman) at 250,000 × g for 18 hr at 4°C, 0.4 ml of fractions
were collected in order from the bottom. To concentrate the sample, 100 µl of 30%
trichloroacetic acid was added to each fraction, mixed, and allowed to stand on ice for 30
min. The mixture was centrifuged at 15,000 × g for 10 min at 4°C, and 500
µl of acetone was added to the precipitate and mixed. After centrifugation at 15,000 ×
g for 10 min at 4°C, the supernatant was removed, and the pellet was
suspended in PBS after complete evaporation of acetone.
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