The substitution of the native all-trans-retinal chromophore of GtACR1 with all-trans-[15-13C,15-2H]retinal and all-trans-[14,15-2H2]retinal followed similar procedures described previously for CaChR1.11 (link) Reconstituted GtACR1 membranes were suspended in 100 mM hydroxylamine buffer with 300 mM NaCl and 20 mM HEPES (pH 7) and exposed to 530 nm light-emitting diode (LED) illumination (5 mW/cm2) for 40 min. Bleaching was monitored by UV–visible spectroscopy using a Cary 6000 spectrometer (Agilent Technologies, Inc., Santa Clara, CA). After >95% conversion of the retinal chromophore to retinal oxime, the sample was pelleted using a SCILOGEX D3024 centrifuge spun at 21000g for 3 min and resuspended in wash buffer [300 mM NaCl and 20 mM HEPES (pH 7)]. This was repeated at least three times to remove unreacted/excess hydroxylamine and free retinal oxime. A 2-fold stoichiometric excess of the isotope-labeled retinal was then added as a 2 mM EtOH solution. The incorporation of the isotope-labeled retinal was verified by resonance Raman spectroscopy (RRS) using a Renishaw inVia confocal Raman microscope with 785-nm laser excitation similar to measurements previously reported.10 (link)
D3024
Manufactured by Scilogex
Sourced in United States
The D3024 is a laboratory-grade centrifuge designed for general-purpose applications. It features a brushless DC motor and a microprocessor-controlled speed and timer. The centrifuge can reach a maximum speed of 4,000 RPM and can accommodate a variety of sample tubes and rotors.
Automatically generated - may contain errors
7 protocols using d3024
1
Isotope-Labeled Retinal Substitution in GtACR1
2
Solubility of FNS in Various Oils
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To determine the FNS solubility in different oils, an excess amount of the drug was added into a separate capped glass vial containing 2 mL of oil (ie oleic acid, olive oil and eucalyptus oil). The mixtures were vortex for 5 min and placed in an isothermal shaking water incubator (WNB14; Memmert GmbH+Co. KG, Germany) at 37 °C for 72 h to maintain equilibrium. All the samples were centrifuged (SCILOGEX, D3024) at 10,000 rpm for 10 min. Finally, the amount of FNS was determined by UV/visible spectrophotometer (UV-1800 240 V; Shimadzu Corporation, Japan) at a wavelength (λmax) of 210 nm using a previously constructed calibration curve in acetate buffer solution (pH 5.5).
3
Quantitative Analysis of Drug-Loaded Solid Lipid Nanoparticles
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The drug content and entrapment efficiency were determined using
a UV spectrophotometer. Exactly 1 gram of SLNs was taken in an Eppendorf
tube (Cat no 007103; SCILOGEX) and centrifuged (D3024, SCILOGEX) at
13 000 rpm for 15 min. The supernatant (0.5 mL) was suitably
diluted with phosphate buffer pH 7.4. The sample was then stirred
for 10 min at 1000 rpm with a magnetic stirrer. The absorbance was
recorded at ƛmax of 248 nm31 (link) using a UV spectrophotometer (UV-1601, SHIMADZU, Japan).
The
sediment was mixed with 1 mL of methanol and vortexed for 5 min to
extract the entrapped drug from the SLNs. It was diluted with phosphate
buffer (pH 7.4) and further stirred for 10 min. The absorbance was
recorded at ƛmax of 248 nm using a UV–visible
spectrophotometer. The drug content which is the quotient of drug
concentration with respect to the total amount of drug-loaded SLNs
is calculated as the sum of the drug in both supernatant and sediment.32 (link) The drug entrapment and association efficiencies
were calculated usingeqs 6 and 7 , respectively.33 (link)
a UV spectrophotometer. Exactly 1 gram of SLNs was taken in an Eppendorf
tube (Cat no 007103; SCILOGEX) and centrifuged (D3024, SCILOGEX) at
13 000 rpm for 15 min. The supernatant (0.5 mL) was suitably
diluted with phosphate buffer pH 7.4. The sample was then stirred
for 10 min at 1000 rpm with a magnetic stirrer. The absorbance was
recorded at ƛmax of 248 nm31 (link) using a UV spectrophotometer (UV-1601, SHIMADZU, Japan).
The
sediment was mixed with 1 mL of methanol and vortexed for 5 min to
extract the entrapped drug from the SLNs. It was diluted with phosphate
buffer (pH 7.4) and further stirred for 10 min. The absorbance was
recorded at ƛmax of 248 nm using a UV–visible
spectrophotometer. The drug content which is the quotient of drug
concentration with respect to the total amount of drug-loaded SLNs
is calculated as the sum of the drug in both supernatant and sediment.32 (link) The drug entrapment and association efficiencies
were calculated using
4
Spectrophotometric Quantification of SLN Drug Encapsulation
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The drug content and encapsulation efficiency of the SLN dispersions was investigated spectrophotometrically. Briefly, 1.5 mL of the sample was added to centrifuge tubes and centrifugated at 14,000 rpm for 10 min using a centrifuge machine (D3024, SCILOGEX, Rocky Hill, CT, USA). The supernatant layer was analyzed with a UV spectrometer (UV-160, Shimadzu, Kyoto, Japan) for free drug content after appropriate dilution with phosphate buffer pH 7.4 at λ max 294 nm. The entrapped drug in the SLN droplets was determined by first dissolving the sediment layer in ethanol followed by dilution and vortex mixing for 10 min [50 (link)]. The encapsulation efficiency of prepared SLNs was then calculated as mentioned in Equation (1):
5
Stability Assessment of Optimized Lipid-Rich Nanoparticle Formulations
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The optimized formulations of both LRX-NE as well as C-LRX NE were investigated under stress conditions for 28 days as per ICH guidelines to analyze their thermodynamic stability profiles. The protocol of this study was followed as per the previously reported study with some modifications as follows [18 (link)]:
The optimized NE formulations containing LRX alone as well as chitosan were initially placed in the incubator at 40 °C for 28 days, followed by their cooling to room temperature. This test was performed to observe the formulation’s physical appearance as well as any evidence of creaming or turbidity.
This test involved subjecting optimized formulations with and without chitosan to centrifugation (D3024, SCILOGEX, Rocky Hill, CT, USA) at a speed of 5000 rpm for 10 min and checking for any visible signs of phase separation.
The optimized LRX formulations with and without chitosan were assessed by freeze–thaw cycle method for 28 days by placing them in a deep freezer (2 °C), followed by holding them at room temperature. This extreme treatment was performed to note whether the formulations would return to their original form or not.
The optimized NE formulations containing LRX alone as well as chitosan were initially placed in the incubator at 40 °C for 28 days, followed by their cooling to room temperature. This test was performed to observe the formulation’s physical appearance as well as any evidence of creaming or turbidity.
This test involved subjecting optimized formulations with and without chitosan to centrifugation (D3024, SCILOGEX, Rocky Hill, CT, USA) at a speed of 5000 rpm for 10 min and checking for any visible signs of phase separation.
The optimized LRX formulations with and without chitosan were assessed by freeze–thaw cycle method for 28 days by placing them in a deep freezer (2 °C), followed by holding them at room temperature. This extreme treatment was performed to note whether the formulations would return to their original form or not.
6
Standardized Arthrocentesis Procedure
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All diagnostic arthrocentesis procedures were performed in the outpatient operating room of our hospital, and all operating steps followed the standardized anatomic positioning procedure. After arthrocentesis, we divided the harvested joint fluid into two portions, one of which was put into a normal centrifuge tube by using a syringe and centrifuged as recommended by Aggarwal et al. [2 (link)] (6600 rpm, 180 seconds; D3024, SCILOGEX, Pittsburgh, PA, USA). For one patient, the sample was not centrifuged because the collected synovial fluid was less than 1.5 mL. Of the 84 samples before centrifugation and the 83 samples after centrifugation, most of the LE strips gradually deepened in color over time after sample application.
7
Microfluidic Screening of P. aeruginosa and L. monocytogenes Responses to Antimicrobial Peptide
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P. aeruginosa and L. monocytogenes treated with IKAFKEATKVDKVVVLWTA (long chain) peptide were used for all microfluidic experiments. The cell suspensions were prepared from overnight cultures grown at 37 °C in their respective broth solutions. One ml of this culture was centrifuged (SciLogex D3024, USA) at 3000 rpm for 3 min. The supernatant was removed, and the pellet was suspended in sterile PBS. This was then diluted to a 0.5 McFarland Standard and used for further experimentation to ensure that cells would be poised to grow once inoculated. The cells were injected into the bacterial dispensing chamber at the center of the device. Once the loading channels and 24 chambers started to fill with bacteria, soy peptide at a concentration of 4.6 µM, 37.2 µM, 74.4 µM, and 298 µM was injected into the inlets of 4 CGGs, along with PBS buffer, using 1 mm syringes with 0.3 mm needle tips (Becton, Dickinson and Company, USA). The flow rate (20 µl/h) was controlled using syringe pumps (Chemyx Fusion Touch 200 and 400, USA; Harvard Apparatus Pump 11 Elite, USA). The temperature of the device was maintained at 37 °C using a temperature controller stage. Once the concentration gradients start to fill and the 24 wells were filled with the peptide and bacterial mixture, the syringe pumps were turned off and the bacteria were incubated in the device.
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