NTX concentration in the skin was quantified upon completion of the in vitro skin permeation studies. The diffusion area was isolated, blotted dry, and then tape-stripped twice to remove residual formulation from the skin surface (Scotch ½” Magic Tape, 3M, Maplewood, MN, USA). The skin was weighed, cut into small pieces, and extracted in methanol overnight under agitation. Each sample was further sonicated for 20 min, centrifuged (AccuSpin Micro 17R, Fisher Scientific, Hampton, NH, USA), appropriately diluted, and syringe-filtered. NTX content was quantified using HPLC. Experiments were performed in triplicate.
Accuspin micro 17r
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AccuSpin Micro 17R is a refrigerated, high-speed microcentrifuge designed for a wide range of applications. It features a compact and ergonomic design, and can accommodate up to 24 microcentrifuge tubes. The unit offers precise temperature control and high-speed performance to meet the needs of various laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
1510 ultrasonic cleaner, by Emerson (2 mentions)
Bead mill 4, by Thermo Fisher Scientific (2 mentions)
Centrifuge 5804 r, by Eppendorf (2 mentions)
Abi 7500 real time pcr instrument, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Precision shallow form shaking water bath, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Sybr premix ex taqtm 2 tli rnaseh plus kit, by Takara Bio (1 mentions)
Th homogenizer, by Omni International (1 mentions)
Sybr premix ex taq 2 tli rnaseh plus kit, by Takara Bio (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest, by BD (1 mentions)
Lc system lc 20ad, by Shimadzu (1 mentions)
5500 qtrap triple quadrupole mass spectrometer, by AB Sciex (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Oasis hlb 96 well plate, by Waters Corporation (1 mentions)
Phos select iron affinity gel, by Merck Group (1 mentions)
Urea pellets, by Merck Group (1 mentions)
Microcon 30 kda centrifugal filter, by Merck Group (1 mentions)
16 protocols using accuspin micro 17r
1
Quantifying NTX Skin Permeation
2
Characterization of Chitosan-NTX Microspheres
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Encapsulation efficiency was determined by dissolving a known weight of chitosan-NTX microspheres in 20 mL of 1% v/v acetic acid under magnetic stirring for 24 h. The solution was centrifuged (AccuSpin Micro 17R, Fisher Scientific, Hampton, NH, USA), syringe-filtered (0.45 µm, EMD Millipore, Burlington, MA, USA), and NTX content was quantified using a validated HPLC method, detailed later in the Methods section. Encapsulation efficiency was determined according to Equation (2), and drug loading capacity was determined according to Equation (3):
where Wexperimental is the mass of NTX recovered from a known weight of chitosan-NTX microspheres, Wtheoretical is the mass of NTX expected in an equivalent weight of chitosan-NTX microspheres, NTXw is the mass of NTX recovered from a known weight of chitosan-NTX microspheres, and MSw is the known weight of chitosan-NTX microspheres. Encapsulation efficiency and drug-loading capacity were determined in triplicate.
where Wexperimental is the mass of NTX recovered from a known weight of chitosan-NTX microspheres, Wtheoretical is the mass of NTX expected in an equivalent weight of chitosan-NTX microspheres, NTXw is the mass of NTX recovered from a known weight of chitosan-NTX microspheres, and MSw is the known weight of chitosan-NTX microspheres. Encapsulation efficiency and drug-loading capacity were determined in triplicate.
3
Quantifying Skin Drug Retention
4
hMSC RNA Isolation and qRT-PCR Analysis
5
Quantifying Osteogenic Gene Expression
6
Immunoprecipitation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IP was performed as described18 (link) with few modifications. Cultured transfected cells grown in 60-mm dishes were harvested 48 h post-transfection and were lysed in cold buffer containing 50 mM Tris (pH 7.8), 0.5% NP-40, 150 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1 mM PMSF, 10 mM sodium butyrate and protease inhibitor mixture on ice for 15 min. All steps were done at 4 °C. Lysates were cleared by centrifugation at maximum speed (accuSpin Micro 17R; Fisher Scientific) for 10 min. An aliquot was kept for analysis as “input”. Lysates were incubated with primary antibodies overnight at 4 °C with rotation; following incubation with Protein A/G-agarose beads (1:1 mix, 30 µL for each) for 1 h. Beads with immune complexes were pelleted by low centrifugation for 5 min and washed 3 times with above cold buffer. Proteins were analyzed by SDS-PAGE followed by western blotting.
7
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed in PBS, trypsinized, and collected by centrifugation at 1000 rpm for 10 min (accuSpin Micro 17R; Fisher Scientific). Cells resuspended in PBS were fixed in cold 70% ethanol. Cells collected by centrifugation at 1500 rpm for 10 min were stained with 50 µg/ml propidium iodide (Sigma) and treated with 100 µg/mL RNaseA for 20 min in the dark. Equal numbers of cells (10 000–30 000) were subjected to flow cytometry at Flow Cytometry Core Facility (BUSM; http://www.bu.edu/cores ) using either BD FACSCalibur or FACScan flow cytometer (BD Biosciences) and was analyzed using either CellQuest (BD Biosciences) or FlowJo (FlowJo LLC).
8
Quantification of Reaction Products by LC-MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reaction products derived were quantified by liquid chromatography-mass spectrophotometry (LC-MS). Firstly, samples withdrawn from the reactor were filtered through a 20-25 μm filter (NO 541 Hardened Ashless, and 110 mm diameter, WhatmanTM Co., Marlborough, MA, USA) to remove the spent catalyst. Then, the filtered solution was diluted 10-fold with HPLC grade water, followed by centrifugation using ultracentrifugation filters (Amicon® filters, Merc Millipore, Billerica, MA, USA) at 9000 rpm for 10 min (accuSpin Micro 17R, Fisher Scientific, Waltham, MA, USA). After centrifugation, the precipitate was discarded, while the supernatant was used for analysis. An aliquot of 10 µL from the supernatant was injected into a Shimadzu LC system (LC-20AD, Shimadzu Corp, Kyoto, Japan), combined with a Qtrap 5500 triple quadrupole mass spectrometer (AB Sciex, Foster City, CA, USA). Analytes were eluded by means of an HPX-87C column (250x4.0 mm, Bio-Rad Aminex, Hercules, CA, USA) operated at 80 °C with a mobile phase consisted of acetonitrile: water (20:80 v/v), at a flow rate of 0.2 mL/min. Mass spectrophotometer analysis was operated in negative mode at a temperature of 500 °C, a curtain gas of 30 psi, an ion source gas of 15 psi for nebulizer (GS1), and heater (GS2). Samples were quantified according to HPLC-grade analytical standards.
9
Histone Extraction and Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histone extraction was done as described earlier.17 (link),18 (link) Cultured cells were lysed with cold 10 mM Tris buffer (pH 8), containing 0.6% NP-40, 0.15 M NaCl, 1 mM EDTA, protease inhibitors (Roche Pharmaceuticals), and 5 mM sodium butyrate. After a 5 min incubation on ice, nuclei were pelleted at 1200 × g (accuSpin Micro 17 R; Fisher Scientific) at 4 °C, and re-suspended in 150 μL 0.4 N H2SO4 on ice. After 20 min of incubation, nuclear debris was removed by centrifugation at 13 000 × g for 10 min at 4 °C. The supernatant was treated with 1.5 mL of cold 20% trichloroacetic acid for 10 min at 4 °C. Pellets were separated by centrifugation at 13 000 × g at 4 °C, and rinsed with 0.1% HCl in acetone and, subsequently, with acetone. The final pellet was re-suspended in reducing SDS-sample buffer.
10
Comprehensive Proteomics Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
○ Urea pellets (Sigma, #U1250)
○ Microcon-30 kDa Centrifugal filters (Millipore, #MRCF0R030)
○ 1.5 ml and 2 ml protein-low-binding tubes
➢ e.g., Eppendorf ProteinLobind tubes (Eppendorf, #022431081 and #022431102)
○ Oasis HLB 96-well μElution plate (2 mg sorbent per well, Waters #186001828BA) for proteomic samples and Oasis HLB 96-well plate (10 mg sorbent per well, Waters #18600128) for phosphoproteomic samples
○ Empore™ SDB-RPS extraction disc (3 M, #00051115088162)
○ Empore™ C8 extraction disc (3 M, # 00051115088049)
○ PHOS-Select™ iron affinity gel (Sigma, #P9740)
○ Tools to cast SDB-RPS and C8 disks onto a pipette tip
➢ e.g. 16 gauge, Kel-F Hub NDL, 2 in, point style 3 needle (Hamilton, #90516) and plunger assembly (Hamilton, #1122-01)
○ 1.5 ml tube holder (GL Sciences, Inc. #5010-21514)
○ Thermal shaker
➢ e.g. Thermomixer C (Eppendorf, #5382000015)
○ 1.5 ml-tube centrifuges
➢ e.g. AccuSpin Micro 17R (Fisher Scientific, #13100676) for FASP
➢ e.g., Rotina 380R (Hettich, #1706-01) for StageTip spinning
○ 96-well plate centrifuge
➢ e.g. Universal 320R (Hettich, #1406-01)
○ Tube rotator
➢ e.g. SB3 (Stuart, no code provided)
○ Equipment for the determination of peptide concentration
➢ e.g., Nanodrop spectrophotometer ND-1000 (Nanodrop Technologies, Inc.)
○ Speed vacuum lyophilisator
➢ e.g., CentriVap centrifugal vacuum concentrator (LabConco, #7810033) and CentriVap cold trap (LabConco, #7385030)
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!