Chimeric VLPs
Paul Pumpens in Single-Stranded RNA Phages, 2020
As mentioned briefly in the Dyed phage particles section of Chapter 6, the phage MS2, but not MS2 VLPs, was labeled at that time with (i) fluorescein-5-isothiocyanate (FITC) (ii) fluorescein, (iii) 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF), and (iv) rhodamine B (Gitis et al. 2002a). The FITC and DTAF were used for the conjugation of lysine residues. The rhodamine B and fluorescein labeling were performed by using 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (DEC) together with the dye. This procedure resulted in permanent attachment without covalent conjugation of the dye, probably due to DEC-assisted caging of the fluorescent labels in the hydrophobic environment of the dye. The fluorescent phage MS2 was introduced into ecological studies as a new tracer for the investigation of pathogen transport in porous media (Gitis et al. 2002b).
Assay for Catalytic Trace Copper in Biological Fluids
Robert A. Greenwald in CRC Handbook of Methods for Oxygen Radical Research, 2018
For each sample, prepare a blank to correct for other TBA-reactive materials which might be present in biological samples, omitting the phenanthroline but adding 0.1 mℓ Chelex resin-treated water. Establish a standard curve for copper concentrations ranging from 1 to 10 μmol/ℓ using cupric chloride. After mixing, incubate the tubes at 37°C for 1 hr in a shaking water bath. Add 0.1 mℓ of 0.1 mM EDTA to each tube to stop the reaction, followed by 0.5 mℓ thiobarbituric acid (1% w/v in 50 mM NaOH) and 0.5 mℓ 28% w/v trichloroacetic acid. Transfer the contents of each tube to a clean glass tube and heat for 10 min at 100°C to develop TBA reactivity. Cool the solution and extract into 3.0 mℓ butan-1-ol by vortex mixing. Separate the phases by centrifugation at 2000 × g for 5 min and measure fluorescence of the butanol phase at 553 nm with excitation at 532 nm. Fluorescence units are expressed relative to a 3 μmol/ℓ standard of rhodamine B as previously described.14
Scanning Angle Interference Microscopy (SAIM)
Qiu-Xing Jiang in New Techniques for Studying Biomembranes, 2020
Variations of intensity in laser illumination in wide-field microscopy could lead to interference fringes. To quantify such variation in intensity, reference slides with a monolayer of fluorescent dye can be prepared using silane conjugates of fluorescein and rhodamine B. The conjugates can be synthesized according to the protocol described in Paszek et al.22 Briefly, 1 mg of fluorescein isocyanate or 1.37 mg of rhodamine B isocyanate is made to react with 9.25 mg of APS in 1.25 mL anhydrous ethanol, under nitrogen gas at ~21°C for 2 hours with constant stirring. Immediately following the reaction, the conjugates are centrifuged at 20,000 g for 10 min to pellet unwanted aggregates, and then diluted 10-fold in absolute ethanol. Clean glass-bottom dishes (#1.5 glass) are incubated with the diluted silane conjugates for 30 minutes at ~21°C, rinsed with water and stored in water until imaging.
Effects of a sub-minimum inhibitory concentration of chlorhexidine gluconate on the development of in vitro multi-species biofilms
Published in Biofouling, 2020
Yuki Suzuki, Tatsuya Ohsumi, Toshihito Isono, Ryoko Nagata, Taisuke Hasegawa, Shoji Takenaka, Yutaka Terao, Yuichiro Noiri
In order to evaluate the entire biofilm structure after being incubated for 6 days, the biofilms were stained with 5 µg ml−1 of Rhodamine B (Wako pure Chemicals, Osaka, Japan) for 5 min at room temperature in the dark, as previously described (Ohsumi et al. 2015), and then observed using a CLSM. Rhodamine B is a counterstaining dye that reveals the amount of biomass of a substance independent of its activity (Rani et al. 2007). Stacks of the fluorescent images were collected and digital reconstruction of the images was conducted by the methods described (Rani et al. 2007). In order to quantify the bio-volumes of bacterial cells and their matrix, the total number of red pixels of the samples in the five fields were enumerated using MetaMorph image analysis software, as described previously (Takenaka et al. 2016).
Evaluation about wettability, water absorption or swelling of excipients through various methods and the correlation between these parameters and tablet disintegration
Published in Drug Development and Industrial Pharmacy, 2018
Baixue Yang, Chen Wei, Yang Yang, Qifang Wang, Sanming Li
MCC as the filler was supplied by Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Mannitol obtained from Beijing Fengli Jingqiu Commerce Co., Ltd. (Beijing, China) was used as excipient and flavoring agent. Crospolyvinylpyrrolidone (PVPP, Kollidon CL) was purchased from BASF AG (Ludwigshafen, Germany). Low-substituted hydroxypropyl cellulose (L-HPC) was provided by Sunshere Pharmaceutical Excipient Co., Ltd. (Anhui, China). Carboxymethyl starch sodium (CMS-Na) was a gift from Huzhou Zhanwang Pharmacy Co., Ltd. (Zhejiang, China). Croscarmellose sodium (CCMC-Na) was obtained from Eli Lilly and Company (Shanghai, China). PVPP, L-HPC, CMS-Na and CCMC-Na were all used as superdisintegrants. Magnesium stearate (MgSt) was purchased from Tianjin Chemical Reagent Co., Ltd. (Tianjin, China) and used as lubricant and glidant. Rhodamine B was supplied by Tianjin Bodi Chemical Co., Ltd. (Tianjin, China). Loratadine provided by Nanjing Zhuo Pu Biotechnology Co., Ltd. (Nanjing, China) was selected as model drug. In the capillary rise test, 97% pure n-hexane was used as the completely wetting liquid. Redistilled water was produced by our laboratory.
Determining the effect of ocular chemical injuries on topical drug delivery
Published in Drug Delivery, 2021
Ghazala Begum, Thomas Leigh, David Stanley, Ann Logan, Richard James Blanch
Porcine corneal disks in inserts placed in wells of Millipore plates were injured with 1 M NaOH, 1 M H2SO4, or HBSS control as described above. After the injury and wash to neutral pH, 30 µL of 1% sodium fluorescein (Sigma-Aldrich, Gillingham, UK) and 50 µg.mL−1 rhodamine B (Sigma-Aldrich, Gillingham, UK) were applied to the corneal epithelial surface such that n = 3 for each application of HBSS-, 1 M NaOH-, and 1 M H2SO4-treated cornea. The Millipore plates were sealed with parafilm and incubated at 37(±1) °C and 5% CO2 for 60 min. The plate inserts with their cornea were carefully removed and the level of rhodamine B and fluorescein that had penetrated through the cornea into the underlying HBSS was measured in the wells of the black-welled plates using the FLUOstar® Omega (BMG Labtech, Aylesbury, UK) and the Infinite® M nano (Tecan, Reading, UK) microplate readers, respectively. For fluorescent analysis, the excitation and emission filters were set at; λexc=485 nm, λemm=520 nm for fluorescein, and λexc=544 nm, λemm=620 nm for rhodamine B, and absorbance measurements using the excitation wavelengths. Point values had the baseline of HBSS-only wells removed.
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