Cells were plated at a concentration of 1.0 × 105 per well (6-well plates) and grown for 24 h in 2 mL of DMEM containing 10% FBS. DMEM (Sigma, D2902, 2 mL) lacking FBS was then added and allowed to condition the media for 24 h. Conditioned media was then collected and distributed into a 96-well assay plate preloaded with 15 μL of a stock solution containing NFF-3 (100 μM in DMSO). Conditioned or control media (300 μL) was added to each well (final concentration NFF-3 = 4.76 μM, MW = 1675.8 g/mole) using a standard 96 well plate. MMP3 activity was monitored at 37°C using a multi-well plate fluorimeter (SpectraMax M2, Molecular Devices). The proteolytic activity of C6/C6-13 conditioned media was measured relative to the background fluorescence produced by unconditioned DMEM. Wavelengths for excitation (325 nm) and fluorescence emission (393 nm) were measured every 10 min over 6 h.
D2902
Manufactured by Merck Group
Sourced in United States
D2902 is a laboratory equipment product. It is a general-purpose device used for various applications in scientific research and analysis. The core function of D2902 is to perform specific tasks required in a laboratory setting. No further details about its intended use or capabilities are provided.
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Lab products found in correlation
Spectramax m2, by Molecular Devices (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Iloprost, by Cayman Chemical (1 mentions)
Hepes, by Merck Group (1 mentions)
Spe confocal microscope, by Leica (1 mentions)
Lumicycle, by Actimetrics (1 mentions)
Picmorg50, by Merck Group (1 mentions)
Dmem hg, by Merck Group (1 mentions)
Ara c, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Sh30031fs, by Thermo Fisher Scientific (1 mentions)
Lumicycle analysis software, by Actimetrics (1 mentions)
Lumicycle 96, by Actimetrics (1 mentions)
Beetle d luciferin, by Promega (1 mentions)
F7524, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Ln229, by American Type Culture Collection (1 mentions)
Celltiter 96 aqueous solution cell proliferation assay, by Promega (1 mentions)
14 protocols using d2902
1
Quantifying MMP3 Activity in Conditioned Media
2
Comparison of DMEM media effects on HeLa cells
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Firstly, the DMEM powder (D2902) was purchased from Sigma-Aldrich and dissolved in sterilized water to form the DMEM medium. Then, DMEM +10% FBS, 25 mM NaHCO3/DMEM +10% FBS, 25 mM HEPES/DMEM +10% FBS, 25 mM pCBMA/DMEM +10% FBS and 50 mM pCBMA/DMEM +10% FBS were prepared and sterilized by passing through a 0.45 μm polyethersulfone membrane filter (Beyotime). All the pH was adjusted to 7.20 using NaOH or HCl solution before adding to the culture flask. Then, 4 mL of Hela cells (50 000 per mL) were cultured in sealed culture flasks over 24 h, and the medium were replaced to corresponding DMEM media, respectively. At the time point, the media were taken out and the pH was tested using pH meter. Cell morphology was captured by a microscope (Nikon, ECLIPSE Ti).
3
Examining PGI2 Effects on Myometrial Gene Expression
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In order to study the effect of PGI2 on the abundance of mRNA transcripts of selected genes, myometrial explants were used. Briefly, a 10 to 20 cm long fragment was cut off from each of six uterine horns, which were transported to the laboratory, and opened longitudinally. Then, the myometrial tissue was separated from the endometrium using scissors, washed with sterile PBS, and cut with a scalpel blade into small pieces (20 to 25 mg). A total of 110–120 mg of the myometrial tissue was placed into glass vials (5 explants per vial) containing 2 mL of incubation medium (Dulbecco’s Modified Eagle’s Medium [D2902, Sigma-Aldrich] supplemented with antibiotics and 25 mM HEPES [H4034, Sigma-Aldrich]) and pre-incubated for 2 h. Then, the pre-incubation media were removed, and myometrial explants were incubated for 8 h in an incubation medium enriched with 0.1% BSA (81-003-3, Millipore, Kankakee, IL, USA) with vehicle (0.01% ethanol; control) or with 1 µM of iloprost (a stable analog of PGI2; 18215, Cayman Chemicals, Ann Arbor, MI, USA). After incubation, myometrial explants were washed with ice-cold PBS, snap-frozen in liquid nitrogen, and maintained at −80 °C until RNA isolation. The treatment was carried out in duplicate using myometrial tissue from six gilts.
4
Schwann Cell Migration in Nerve Regeneration
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Rat pups were obtained from the Plymouth Medical School Animal Facility and euthanized by cervical dislocation. Rat Schwann cells were prepared from the sciatic nerve and brachial plexus of 3-day-old Sprague-Dawley rat pups. Mouse Schwann cells were cultured from the distal sciatic nerve 7 days after transection injury (Woodley et al., 2019). Tibial nerves from GFP-positive control or Runx2 KO mice were cut into 2-mm pieces in a petri dish with a surgical blade. Nerve explants were transferred to 24-well plates and cultured in 1 mL low-glucose (1 g/mL) DMEM (D2902, Sigma) containing 10% FBS for 150 hours. GFP-positive migrating Schwann cells from tibial nerve explants were imaged using a LeicaIM8 fluorescence microscope (Leica, Wetzlar, Germany). Migrating Schwann cells were counted after the images were taken. In vivo Schwann cell migration was assessed at day 6 following sciatic nerve transection injury in both GFP-positive control and Runx2 KO mice. On day 6, transected sciatic nerves were harvested and fixed in 4% paraformaldehyde (in PBS, PH7.2, Sigma) at 4°C overnight. Nerves were cleared with 25%, 50%, and 75% glycerol (# 56-81-5, Sigma, in PBS) for imaging with a LeicaSPE confocal microscope (Leica). Z-stack images were taken and combined to measure the area of migrating Schwann cells (GFP-positive) in the nerve bridge.
5
Bioluminescent Circadian Rhythm Monitoring
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Control and SCN-DKO mice heterozygous for PER2::LUC were housed on 12:12 LD before brain extraction 2 to 4 hours before lights off. SCN slices were made at 200-μm thickness in cold slicing medium [1× Hanks’ balanced salt solution, penicillin-streptomycin (100 U/ml), sodium bicarbonate (375 μg/ml), and Hepes (2.38 mg/ml)] and cultured on membranes (Millipore, PICMORG50) in 35-mm dishes with 1.2 ml of culture medium [Dulbecco’s Modified Eagle’s medium (Sigma-Aldrich, D2902), penicillin-streptomycin (25 U/ml), sodium bicarbonate (352.5 μg/ml), Hepes (2.38 mg/ml), and d -glucose (3.5 mg/ml)]. For every 6 ml of culture medium, 6 μl of 0.1 M luciferin and 120 μl of 50X B-27 Plus were added. Slices were incubated in LumiCycle (Actimetrics) at 37°C. Luminescence counts were made for each sample every 10 min for 14 days, with a medium change on the seventh day. Luminescence traces were normalized by subtracting a 24-hour running mean (center-aligned) baseline. Period was measured by Lomb-Scargle periodogram on the normalized traces following the first medium change (days 8 to 14).
6
Primary Culture of Hypothalamic Neurons
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Primary cultures of hypothalamic neurons were prepared from E18 embryos from pregnant Sprague-Dawley rats. Embryos were euthanized by decapitation, and the hypothalami were dissected free of extraembryonic membranes using a magnifying glass. Then the hypothalami were digested using trypsin-EDTA in HBSS (10 min, 37 °C). Tissues were washed and dissociated using high-glucose Dulbecco’s modified Eagle’s medium (HG-DMEM; D2902, Sigma-Aldrich) supplemented with horse serum (adhesion medium) by pipetting with a Pasteur pipet. The resulting cell suspension was sifted using two cell strainers (70 and 40 μm). Then the cells were seeded on poly-L-lysine-covered vessels. After a 2-h incubation in the adhesion medium, the media was changed to neurobasal medium (21103049, Gibco, USA) supplemented with B27 (17504044, Gibco), L-glutamine (SH30034.01, Hyclone, Logan, Utah, USA) and penicillin/streptomycin (15140122, Gibco). Half of the medium was changed after three days, followed by a complete medium change every 3 days until day in vitro (DIV) 10. From DIV2, 3 µM AraC (C6645, Sigma-Aldrich) was added to cultured hypothalamic cells, to inhibit uncontrolled proliferation of non-neuronal cells [77 (link)].
7
Deuterated Cell Culture Medium Preparation
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Deuterated cell culture medium (DMEM-d) was prepared by dissolving
powdered DMEM cell culture medium (1000 mg/L glucose andl -glutamine, without sodium bicarbonate and phenol red; D2902-Sigma-Aldrich)
in D2O (10 mg/mL, according to the manufacturer’s
instructions). The solution was treated with Me2SO2 (ca. 6 × 10–3 M), NaH2PO4, and Na2HPO4 (25 mM total phosphate,
pD = 7.4),95 (link) then stored under N2. Solutions of diiron complexes in a DMEM-d/CD3OD 5:2 v/v mixture were prepared and treated as described
above. The residual amount of starting material in solution after
24 h at 37 °C was calculated with respect to Me2SO2 as an internal standard (Table 2 ).
powdered DMEM cell culture medium (1000 mg/L glucose and
in D2O (10 mg/mL, according to the manufacturer’s
instructions). The solution was treated with Me2SO2 (ca. 6 × 10–3 M), NaH2PO4, and Na2HPO4 (25 mM total phosphate,
pD = 7.4),95 (link) then stored under N2. Solutions of diiron complexes in a DMEM-d/CD3OD 5:2 v/v mixture were prepared and treated as described
above. The residual amount of starting material in solution after
24 h at 37 °C was calculated with respect to Me2SO2 as an internal standard (
8
Circadian Rhythm Monitoring in Per2:luc Mice
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ChP explants from Per2:luc mice43 (link) were dissected in HBSS (Fisher, SH30031FS) and maintained on wet ice until plated. Explants were transferred to 24 well plates with 500 μL of filter-sterilized Lumicycle media (phenol-free DMEM (Sigma D-2902), 10 mM HEPES (pH 8.0), 4 mM sodium bicarbonate, 25 mM D-glucose, 1 × B27 (Gibco), 4mM L-glutamine, Penicillin/Streptomycin) freshly supplemented with 100 mM beetle D-luciferin (Promega, E1601) and/or 100 nM dexamethasone. PCR plate sealer was used to seal the wells for the duration of the experiment (ThermoFisher) and placed in a Lumicycle-96 (Actimetrics) in a water-jacketed incubator at 35˚C. Luminometry was performed by iterative measurement in 60 s bins, 10 times per hour. Luminometry data was analyzed with Lumicycle analysis software (Actimetrics). Only traces with a goodness-of-fit of at least 80% were included. Period was measured using the χ2 function.
9
Preparation and NMR Analysis of Ru Complexes in DMEM
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Powdered DMEM
cell culture medium (1000 mg/L glucose andl -glutamine, without
sodium bicarbonate and phenol red; D2902; Sigma-Aldrich) was dissolved
in D2O (10 mg/mL), according to the manufacturer’s
instructions. The solution of deuterated cell culture medium (“DMEM-d”) was treated with Me2SO2 (6.6 × 10–3 M) and NaH2PO4/Na2HPO4 (0.10 M, pD = 7.5)120 −122 (link) and then stored at 4 °C under N2. The selected ruthenium compound (2–3 mg) was dissolved
in DMSO-d6 (0.14 mL; 0.18 mL for4 ) and then diluted with DMEM-d up to 0.75
mL total volume (cRu ca. 4 × 10–3 M). The mixture was stirred for 30 min, then filtered
over celite, and transferred into an NMR tube. The resulting yellow
solution was analyzed by 1H and 31P NMR (delay
time = 3 s; number of scans = 20) and then heated at 37 °C for
24 h. After cooling to room temperature, NMR analyses were repeated.
Compound1 was instead dissolved in a DMSO-d6/DMEM-d 4:1 v/v solution (0.7 mL; [Ru]
= approx. 3 × 10–3 M) containing Me2SO2 as the internal standard.115 (link) The percentage of the starting complex + related aquo complex was
calculated by the relative integral with respect to Me2SO2 (δ/ppm = 3.16 in DMSO-d6/DMEM-d 1:4 and 1:3 v/v; δ/ppm = 2.95
in DMSO-d6/DMEM-d 4:1
v/v).
cell culture medium (1000 mg/L glucose and
sodium bicarbonate and phenol red; D2902; Sigma-Aldrich) was dissolved
in D2O (10 mg/mL), according to the manufacturer’s
instructions. The solution of deuterated cell culture medium (“DMEM-d”) was treated with Me2SO2 (6.6 × 10–3 M) and NaH2PO4/Na2HPO4 (0.10 M, pD = 7.5)120 −122 (link) and then stored at 4 °C under N2. The selected ruthenium compound (2–3 mg) was dissolved
in DMSO-d6 (0.14 mL; 0.18 mL for
mL total volume (cRu ca. 4 × 10–3 M). The mixture was stirred for 30 min, then filtered
over celite, and transferred into an NMR tube. The resulting yellow
solution was analyzed by 1H and 31P NMR (delay
time = 3 s; number of scans = 20) and then heated at 37 °C for
24 h. After cooling to room temperature, NMR analyses were repeated.
Compound
= approx. 3 × 10–3 M) containing Me2SO2 as the internal standard.115 (link) The percentage of the starting complex + related aquo complex was
calculated by the relative integral with respect to Me2SO2 (δ/ppm = 3.16 in DMSO-d6/DMEM-d 1:4 and 1:3 v/v; δ/ppm = 2.95
in DMSO-d6/DMEM-d 4:1
v/v).
10
pH-Controlled Cell Culture Media Preparation
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Culture media at different pH were prepared as described8 (link)60 . Briefly, low glucose DMEM without sodium bicarbonate (Sigma, D2902) was buffered with HEPES (4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid; Sigma, H0887), EPPS (4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid; Sigma, E9502) and MES (2-(N-Morpholino) ethanesulfonic acid; Sigma, M3671) (8mM each) and pH was adjusted with HCl/NaOH. They were kept iso-osmotic by adding NaCl and NaHCO3. The pH stability was monitored at the initiation and completion of the experiments.
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