The NPs loaded with Sal were prepared by a modified single emulsion method. Briefly, 20 mg of each copolymer and 2 mg Sal were dissolved in 1 mL of DCM. The mixture was emulsified in 3 mL of aqueous polyvinyl alcohol (PVA) solution at 3% (w/v) by sonication (XL2000, Misonix, Farmingdale, NY, USA) for 60 s (4 W) to obtain an o/w emulsion. This emulsion was then emulsified in 5 mL of aqueous solution containing 0.5% (w/v) PVA by sonication (XL2000, Misonix, USA) for 10 s (2.5 W). The w/o/w emulsion formed was gently stirred at room temperature in a fume hood until the evaporation of the organic solvent was complete. The resulting solution was filtered to remove nonincorporated drugs. Blank NPs were produced in the same manner without adding Sal.
Xl 2000
Manufactured by Bioventus
Sourced in United States
The XL-2000 is a versatile laboratory equipment designed for various applications. It features a large capacity and precise temperature control, making it suitable for a wide range of sample processing and analysis tasks. The core function of the XL-2000 is to provide a controlled environment for efficient and reliable experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Sw28ti rotor, by Beckman Coulter (2 mentions)
Elution buffer, by Santa Cruz Biotechnology (1 mentions)
Protein g agarose beads, by Merck Group (1 mentions)
Rbpj antibody, by Cell Signaling Technology (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
0.22 m pvdf filter, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
α actin, by Abcam (1 mentions)
Tgf β, by Abcam (1 mentions)
Gapdh, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
T7 express lysy iq competent e coli, by New England Biolabs (1 mentions)
Ni nta column, by Qiagen (1 mentions)
Glycogen assay kit 2, by Abcam (1 mentions)
Strata x polymeric reverse phase column, by Phenomenex (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
50.2 ti rotor, by Beckman Coulter (1 mentions)
21 protocols using xl 2000
1
Salicylic Acid-Loaded Polymeric Nanoparticles
2
RBPJ Chromatin Immunoprecipitation Protocol
3
Folate-Conjugated Polymeric Nanoparticles
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A tetrahydrofuran solution (1 mL) containing CP, DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]), and DSPE-PEG2000-folate (1 mg each) was mixed with water (10 mL), followed by sonicating the mixture for 60 seconds at 12 W output using a microtip probe sonicator (XL2000, Misonix Inc., Farmingdale, NY, USA). The mixture was then stirred at room temperature overnight to evaporate the tetrahydrofuran to yield the final folate-CP dots suspended in water.
4
Intestinal Barrier and Transit Assay
5
Preparation of Oral Bacterial Extracts
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Oral bacteria, including Actinomyces naeslundii NC-3, Streptococcus gordonii DL1, S. cristatus CC5A, S. mutans KPSK2, Actinomyces viscosus EG4, and Porphyromonas gingivalis 33277, which were from our laboratory collection at Meharry Medical College School of Dentistry, were grown in appropriate media aerobically or anaerobically at 37°C for 16 h. Bacterial cells were harvested by centrifugation at 6,000×g for 10 min at 4°C. Bacterial cells were washed twice with cold PBS. The cell extracts were then prepared by sonication at Power 15 (10×30 sec) using a Misonix XL-2000. The supernatants were collected and filtered through a 0.22 µm PVDF filter (Millipore). The bacterial extracts were stored in −20°C freezers if long term storage were needed.
6
Arterial Tissue Protein Extraction
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The following solutions were added to each arterial tissue sample (wet weight ≤30 mg): 300 μl RIPA buffer (Sigma-Aldrich), 3 μl protease inhibitor cocktail (Sigma-Aldrich), 0.5 μl of 50 mM PMSF. Samples were kept at 4°C during the following procedures. Arteries were diced using scissors, sonicated for 3–4 s twice (speed 6; XL2000, Misonix, NY, USA), centrifuged at ~18,000 g for 5 min, and then the supernatant was collected. As per manufacturer's instructions, the protein content of each sample was quantified using Bio-Rad's Dc™ Protein Assay Kit (Bio-Rad Laboratories Pty., Ltd., Gladesville, NSW, Australia). Samples were aliquoted in volumes required to load 20 μg of protein per well on the gels and stored at −80°C.
7
Aortic SMC Protein Expression
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Abdominal aortic SMCs were incubated in six-well plates with vehicle (DMSO, final 0.1 %) or E2 (1–100 nM) for 48 h prior to protein extraction. Extraction was performed with M-PER with protease (Roche-Mini tablets) and phosphatase inhibitors (Thermo Fisher phosphatase inhibitor tablets; 200 μL per well) and samples were sonicated for 10 s on a setting of 10 (Misonix, XL-2000) and then incubated on ice for 30 min. Cell extracts were centrifuged (10,000g′s) for 10 min to pellet cellular debris, and protein concentration was quantified in supernatants (BCA assay; ThermoFischer). For Western analyses, protein (12 μg) was electrophoresed on a 12 % reducing SDS-PAGE gel. Antibodies utilized for Western analyses were α-actin (Abcam, cat#5694), β-actin (Sigma mouse, cat#A5441), SM22α/transgelin (Abcam rabbit, cat#14106), and TGF-β (Abcam rabbit, cat#66043; which recognizes all three isoforms of TGF-β). Protein content was quantified using background subtraction (50 pixels per blot subtracted) followed by the gel plug-in for ImageJ 1.48v and was normalized to GAPDH (Sigma mouse, cat#G9295).
8
Purification of Recombinant Proteins
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The ruvCgfp and ruvCDefgfp genes were subcloned into pET28a expression vectors and then transformed into the BL21 derivative strain (T7 Express lysY/Iq Competent E. coli, New England Biolabs). After induction with 0.1 mM IPTG, the proteins with an N-terminal His6-tag were produced at 20°C for 16 hours and pelleted by centrifugation at 7000 rpm for 30 min. The cell pellet was resuspended in lysis buffer [50 mM Hepes (pH 8.0), 300 mM NaCl, 5 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride] and disrupted by sonication (10 times for 30 s, on/off at output 5; XL-2000, Misonix). The total cell lysate was centrifuged at 30,000g for 40 min, and the soluble recombinant protein was purified by immobilized metal ion chromatography with a Ni-NTA column (Qiagen). After washing with wash buffer (lysis buffer with 50 mM imidazole added), the protein was eluted with elution buffer (lysis buffer with 500 mM imidazole) and further desalted against the storage buffer [20 mM Hepes (pH 8.0) and 20 mM NaCl]. The protein was concentrated to about 2 mg/ml and stored at −80°C. The Flp protein was purified to near homogeneity using DNA affinity enrichment as the final step (81 (link)).
9
Quantification of Cellular Glycogen
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Glycogen was quantified using the Glycogen Assay Kit II (colorimetric) from AbCam (ab169558) according to manufacturer’s instructions. Briefly, approximately 2 x 106 cells were washed with ice-cold PBS and collected via scraping in 300μl ice-cold ddH2O. Cells were immediately sonicated on ice for 5 x 1s pulses with a probe sonicator (Misonix Inc, model XL2000), then heated to 80°C for 10 minutes. Samples were stored overnight at -20°C, then thawed, centrifuged at 12,000 x g for 10 minutes and the supernatants collected and assayed for glycogen hydrolysis and quantification, followed by protein concentration determination (Pierce BCA Protein Assay kit #23227) according to manufacturer’s instructions.
10
DOC-Loaded PEG-PCL Nanoparticle Fabrication
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The DOC-loaded (DOC-PEG-PCL-mAb and DOC-PEF-PCL-IgG) NPs were formulated by a single-emulsion solvent evaporation technique according to previously published work with slight modifications.30 (link) In brief, 5 mg of DOC was dissolved in 0.5 mL of chloroform in a beaker and mixed with 20 mg of mAb-conjugated PEG-PCL. To generate the emulsion, the mixture was added dropwise to 1.5 mL of a 3% (w/v) PVA solution and then sonicated (XL2000; Misonix Inc., Farmingdale, NY, USA) for 1 minute (32.5 W) to obtain an O/W emulsion. This emulsion was then emulsified in 2.5 mL of an aqueous solution containing 0.5% (w/v) PVA by sonication for 30 seconds (26 W). The O/W emulsion was then gently stirred at RT in a fume hood until all the organic solvent evaporated. The resulting solution was filtered through a 0.45-µm microporous membrane to remove non-incorporated drugs. Blank PEG-PCL NPs were prepared in the same manner, but without adding DOC. The fluorescent NPs were prepared in a same way except that the DOC was replaced with Coumarin 6.
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