A 15% (w/v) solution of pluronic gel was made by heating sterile DNase/RNase-free PBS to 90°C and slowly mixing in the pluronic (Sigma) powder. Once all powder was dissolved, the magnetic stirring plate and gel were placed at 4°C until implantation. Immediately prior to surgery, miR-125b mimic (Qiagen) was mixed with the 15% pluronic gel at 4°C to obtain a final concentration of 10 μM.
Pluronic
Manufactured by Merck Group
Sourced in United States, Germany
Pluronic is a type of lab equipment used for a variety of applications. It is a block copolymer composed of polyethylene oxide and polypropylene oxide. Pluronic serves as a surfactant and emulsifier, having the ability to solubilize and disperse various compounds. The core function of Pluronic is to modify the physical and chemical properties of liquids and solutions.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Fatal plus, by Vortech Pharmaceuticals (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Fura 2, by Thermo Fisher Scientific (2 mentions)
Eclipse ti u microscope, by Nikon (1 mentions)
Fura 2 am, by Merck Group (1 mentions)
Candida antarctica lipase b, by Merck Group (1 mentions)
Axioscope, by Zeiss (1 mentions)
Dimethyl sulphoxide, by Merck Group (1 mentions)
Pyrene, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dspe peg, by Nanocs (1 mentions)
Magna pure 96 dna and viral na small volume kit, by Roche (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Facsaria 3, by BD (1 mentions)
29 protocols using pluronic
1
Pluronic Gel Delivery of miR-125b
2
Pulmonary Toxicity of Coated MWCNTs
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Mice (C57BL6, Jackson Laboratories) were exposed to uncoated MWCNTs or Al2O3-coated MWCNTs at 4 mg/kg in 0.1% Pluronic (Sigma-Aldrich) surfactant solution or 0.1% Pluronic alone via oropharyngeal aspiration while under isoflurane anesthesia. Mice were euthanized via intraperitoneal injection of Fatal Plus (Vortech Pharmaceuticals, Dearborn, MI) on day 1 or day 28 after MWCNT exposure. At necropsy, the lungs were lavaged with Dulbecco's Phosphate Buffered Saline (DPBS) and bronchoalveolar lavage fluid (BALF) was collected for cell differential counts and enzyme-linked immunosorbent assay (ELISA). The middle and caudal lobes of the right lung were collected for mRNA analysis and stored in RNAlater as per manufacturer's instructions (Ambion, Austin, TX). The left lungs were intratracheally infused with 10% neutral buffered formalin and fixed for 24 hrs and then transferred to 70% ethanol. The left lung was cross-sectionally cut into three portions, embedded in paraffin and sectioned and stained with either hematoxylin and eosin (H&E) or Masson's trichrome to visualize collagen deposition in the lungs.
3
Measuring Intracellular Calcium Dynamics in TG Neurons
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Changes in intracellular calcium levels were measured using Fura-2AM (Invitrogen) as described [8] (link). Briefly, cultured TG neurons on poly-D-lysine-laminin-coated coverslips were incubated with Fura-2AM (2 μM) for 30 min at 37°C in the presence of 0.05% Pluronic (EMD Millipore, Philadelphia, PA) in a standard extracellular solution (SES) containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, pH 7.4. Nucleofected cells were identified by the presence of GFP. Intracellular calcium accumulation was measured following TRPV1 activation with CAP (50 nM). Fluorescence was detected with a Nikon Eclipse Ti-U microscope fitted with a X20/0.8 Na Fluor objective, and images from 340 and 380 nm excitation wavelengths were collected and analyzed with MetaFluor Software (MetaMorph). The net change in calcium (ΔF340/380) was calculated by subtracting the basal F340/380 ratio from the peak F340/380 achieved during stimulation.
4
Efficient Protoplast Isolation from A. teichomyceticus
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A. teichomyceticus protoplasts were prepared by modifying the method described in [15 (link)] in order to reduce residual contaminating hyphae and increase protoplast number and regeneration efficiency. In brief, ca. 100 mL of growth culture were centrifuged at 3250× g. The mycelium was washed once in P medium [39 (link)], then 10 g (fresh weight) were suspended in 50 mL of P medium. For cell wall digestion, hen egg white lysozyme (HEWL) (Merck KGaA, Darmstadt, Germany) and Candida antarctica Lipase B (Merck KGaA, Darmstadt, Germany) were added at a final concentration of 10 mg/mL and 0.1 mg/mL, respectively. The non-ionic detergent Pluronic (Merck KGaA, Darmstadt, Germany) was supplemented at the final concentration of 0.1 mg/mL. The digestion solution was then incubated at 28 °C with gentle shaking at 50 rpm, for 24 h. Protoplasts were detached from residual mycelium clumps by thoroughly pipetting up and down, then separated from residual hyphal fragments by filtration through 5-μm durapore membrane filters (Merck Millipore, Burlington, MA, USA). The protoplast suspension was then centrifuged at 16,200× g, and finally re-suspended in fresh P medium. The formation of protoplasts was monitored by microscopic observation (Zeiss Axioscope, Carl Zeiss, Jena, Germany) at 400× magnification. The total protoplast number was determined by using a Petroff-Hausser counting chamber.
5
Pluronic-Based Aceclofenac Formulation
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Samples of pluronic (Sigma Aldrich) F108 and L81 with a molecular weight of 14,600 and 2750 g mol−1, respectively, were used with no further purification. Aceclofenac was procured from MMC Healthcare Ltd. Dimethylsulphoxide (DMSO) and Pyrene samples (Sigma Aldrich) were procured. All the reagents were of analytical grade. Triply distilled, water was used for the experiment.
6
Acid-Oxidized MWCNT Nanoparticle Preparation
7
Adenovirus-free AAV Vector Production
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AAV vectors used in this study were produced using a slight modification of
the adenovirus-free transient transfection methods as described
earlier.61 (link),62 (link) Briefly, adherent human embryonic kidney cells
(HEK293) cells grown in roller bottles were transfected with the three
plasmids containing the adenovirus helper proteins, the AAV Rep and Cap
genes, and the ITR-flanked transgene expression cassette. After 72 hours of
transfection, cells were harvested, lysed by sonication, and treated with
benzonase (Merck-Millipore, Darmstadt, Germany). Vectors were then purified
using two successive ultracentrifugation rounds in cesium chloride density
gradients. Full capsids were collected, the final product was formulated in
sterile phosphate buffered saline containing 0.001% of pluronic (Sigma
Aldrich, Saint Louis, MO), and stored at −80°C.
Titers of AAV vector stocks were determined using quantitative real-time
polymerase chain reaction (qPCR). Viral DNA was extracted using the MagNA
Pure 96 DNA and viral NA small volume kit (Roche Diagnostics, Indianapolis,
IN) according to manufacturer’s instructions. The qPCR was performed
in ABI PRISM 7900 HT Sequence Detector using Absolute ROX mix (Taqman,
Thermo Fisher Scientific, Waltham, MA). Specific probe and primers were as
follows: forward 5′-GGCGGGCGACTCAGATC-3′, reverse
5′-GGGAGGCTGCTGGTGAATATT-3′, and probe
5′-AGCCCCTGTTTGCTCCTCCGATAACTG-3′.
the adenovirus-free transient transfection methods as described
earlier.61 (link),62 (link) Briefly, adherent human embryonic kidney cells
(HEK293) cells grown in roller bottles were transfected with the three
plasmids containing the adenovirus helper proteins, the AAV Rep and Cap
genes, and the ITR-flanked transgene expression cassette. After 72 hours of
transfection, cells were harvested, lysed by sonication, and treated with
benzonase (Merck-Millipore, Darmstadt, Germany). Vectors were then purified
using two successive ultracentrifugation rounds in cesium chloride density
gradients. Full capsids were collected, the final product was formulated in
sterile phosphate buffered saline containing 0.001% of pluronic (Sigma
Aldrich, Saint Louis, MO), and stored at −80°C.
Titers of AAV vector stocks were determined using quantitative real-time
polymerase chain reaction (qPCR). Viral DNA was extracted using the MagNA
Pure 96 DNA and viral NA small volume kit (Roche Diagnostics, Indianapolis,
IN) according to manufacturer’s instructions. The qPCR was performed
in ABI PRISM 7900 HT Sequence Detector using Absolute ROX mix (Taqman,
Thermo Fisher Scientific, Waltham, MA). Specific probe and primers were as
follows: forward 5′-GGCGGGCGACTCAGATC-3′, reverse
5′-GGGAGGCTGCTGGTGAATATT-3′, and probe
5′-AGCCCCTGTTTGCTCCTCCGATAACTG-3′.
8
Polymer-Based Organogel Encapsulation
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A polymer-based organogel was prepared by mixing SEBS (molecular weight about 118,000, Sigma-Aldrich) with 1% by weight F68 flake (Pluronic, Sigma-Aldrich) and undecane–hexadecane oil (50:50 by volume) at a concentration of 20 mg ml−1. The mixture was then stirred at 95 °C in a closed vial. Once a clear liquid had been formed, it was cooled to 37–40 °C before use. Organogel encapsulation was conducted by replacing the silicone oil with the molten polymer–oil mixture at the last oil transfer step. Lipid-free organogel (1 ml) was used to wash and cover the droplet power sources. After the transfer to organogel, the encapsulated droplet power sources were moved to a fridge (4 °C) in which the organogel solidified. The final construct was gently extracted from the mould, forming a freestanding droplet power source. Electrodes can pierce through the solidified organogel for measurement of the power output.
9
Fura-2 AM Calcium Imaging Protocol
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Dishes were removed from the incubator, and excess DMEM/F‐12 with 20% FBS and antibiotic antimycotic solution was removed from the glass‐bottomed Petri dish with 1.5 mL of the solution remaining in the dish. Fifteen microlitres of a solution containing 5 µM of Fura‐2 AM dye (cat. no. ab120873, Abcam, Waltham, MA, USA), 70 µM dimethyl sulfoxide (cat. no. 1003427034, Sigma‐Aldrich), and 5 µL of 8 mM Pluronic (cat. no. 102441509, Sigma‐Aldrich) was added into the dish and the dish was gently swirled so the dye was evenly distributed over the fibres before being incubated at 37°C for 30 min. Once finished, the dishes were moved in the dark onto an inverted microscope (CK40, Olympus Life Science, Tokyo, Japan) and continuously superfused via an analog pump (Reglo, Ismatec, Glattburg, Switzerland) with room temperature (∼25°C) experimental Tyrode solution (mM): 121 NaCl, 5.0 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24 NaHCO3, 0.1 EDTA, 5.5 glucose, and ∼0.2% heat‐inactivated newborn calf serum (cat. no. 26010074, Thermo Fisher Scientific), bubbled with 95% O2–5% CO2 giving the bath a pH of ∼7.4 while remaining in the dark.
10
Calcium Imaging of Cell Responses
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Cells were incubated with 1 μM Fluo-4-AM (Molecular Probes-Invitrogen) and 0.01% pluronic (Sigma-Aldrich) in the extracellular solution (NaCl 136 mM, KCl 5.4 mM, MgCl2 1 mM, CaCl2 1.8 mM, HEPES 10 mM, Glucose 10 mM, and NaH2PO4 0.33 mM, pH7.4, osmotic pressure 300 mOsm/L, MgCl2 replaced CaCl2 for calcium free solution) at 25°C for 1 h. The cells were continuously superfused with the extracellular solution and imaged using an inverted microscope (Leca DMI4000B). Drugs (or vehicle control) were applied through a micro-perfusion tube positioned to the vicinity of the cells in the field of view. Fluorescent signal was excited at 510 nm and acquired at 580 nm, and taken every 3 s through a CCD camera. The signal was monitored online and analyzed offline, using Leica AF6000 software (Leica). The fluorescent traces were calculated as:
(F0: the baseline fluorescence of cells before treatments, F: the fluorescence of cells with drugs treatments).
(F0: the baseline fluorescence of cells before treatments, F: the fluorescence of cells with drugs treatments).
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