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Assessment of Airway Smooth Muscle Growth and Division: In Vitro Studies
Published in Alastair G. Stewart, AIRWAY WALL REMODELLING in ASTHMA, 2020
An important limitation of these assay systems is that the colour reaction developed is not only proportional to the cell number, but also to the expression and modulation of the metabolic behaviour exploited by the particular assay. For example, the MTT-reduction assay could give reduced sensitivity compared with cell counts (or 3H-thymidine uptake) in cells of low mitochondrial succinyl dehydrogenase activity or when inhibitors of this enzyme or of mitochondrial function are used.81 Preliminary experiments should therefore be performed to discount such limitations.
Ultraviolet and Light Absorption Spectrometry
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Zoltan M. Dinya, Ferenc J. Sztaricskai
The analytical determination of chloramphenicol is well established, and several colorimetric methods based on spectrophotometry have been described [343—356]. For the color reaction, 2,4,6-trinitrobenzenesulfonic acid was applied by Pietta et al. [348]. Patel et al. [350] showed that chloramphenicol gives a yellow color upon treatment with isonicotinic hydrazine in 30% potassium hydroxide solution. Hassan and Eldesouki [351] reduced the antibiotic in the presence of cadmium, and the reduced product gave yellow-colored complex with the metal. The method of Krzek and Lechniak [347] is also based on complex formation with copper(II) salts in alkaline methanol followed by colorimetric assay at 550 nm. Chloramphenicol was first reduced by Gagne et al. [353] and then treated with p-dimethylaminobenzaldehyde to obtain a colored Schiff base. After the reduction of the antibiotic with zinc, Devani et al. [354] produced a colored complex with Na3FeNH3(CN)5, the specific light absorption of which was found to be suitable for quantitative determination. According to a recent method of Das et al. [356], chloramphenicol is treated with thiourea and the UV absorption of the resulting complex is measured at 340 nm. (These authors noted that the application of thiourea was also successful for the determination of streptomycin.)
Laboratory Procedures and Management
Published in Jeremy R. Jass, Understanding Pathology, 2020
Contrasting with the empirical approach employing natural and synthetic dyes derived from the textile industry is ‘designer’ staining utilising predicted biochemical reactions between specific reagents (reactive agents) and substrates (biochemical components within the cell upon which reagents act). This may be likened to the controlled biochemistry within a test tube except that the reaction (described as histochemistry) occurs within a tissue section. The substrates may include all manner of biological molecules, for example enzymes, carbohydrates, hormones and even DNA. Histochemistry permits the visual demonstration of molecules in their normal location by means of coloured reagents. The intensity of the colour reaction may even indicate the amount of cellular substrate, as well as its exact site of production within the cell.
Modifying antibody-FcRn interactions to increase the transport of antibodies through the blood-brain barrier
Published in mAbs, 2023
Jason Tien, Dmitri Leonoudakis, Ralitsa Petrova, Vivian Trinh, Tetsuya Taura, Debapriya Sengupta, Lisa Jo, Angela Sho, Yong Yun, Eric Doan, Anita Jamin, Hussein Hallak, David S. Wilson, Jennifer R. Stratton
Antibody concentrations in murine serum and CSF were measured with a human IgG ELISA kit (Abcam, #195215) following manufacturer’s standard protocol. In brief, the test antibody was used as a standard to quantify antibody concentration in serum and CSF samples. Standards and test samples were diluted to a concentration below 15 ng/mL and loaded at 50 μL/well into 96-well plates coated with the primary antibody provided by the kit. Kit secondary antibodies were then added to each well, and plates were sealed and incubated at room temperature for 40 min on a plate shaker set at 400 revolutions per minute (rpm). Plates were washed and 3,3‘,5,5’-tetramethylbenzidine was added as a chromogen at 100 μL/well. Sealed plates were incubated at room temperature for 5 min on a plate shaker set at 400 rpm to develop a color reaction, after which the color reaction was stopped. The intensity of the color reaction was assessed by absorbance at a wavelength of 450 nm. Sample protein concentrations were determined by interpolating the blank control absorbance values subtracted against the respective antibody standard curve with a second order polynomial model. PK parameters were calculated using standard noncompartmental methods with Phoenix® WinNonlin® software (Pharsight Corporation, a Certara Company).
Growth hormone improved oxidative stress in follicle fluid by influencing Nrf2/Keap1 expression in women of advanced age undergoing IVF
Published in Gynecological Endocrinology, 2022
Zhaoyan Nie, Na Zhang, Lina Guo, Cuiting Lv, Yi Zhang, Congmin Wang, Haifeng Wu
MDA, GSH-px, CAT and SOD in FF were measured using commercially available kits. The MDA was determined according to the TBA method. One hundred microlitres of test sample and 1,000 μl of working reagent were mixed in a centrifuge tube, and then the standard tube and blank tube were set. The solutions were incubated in boiling water for 40 min. After cooling, the solution was centrifuged at 4,000 rpm for 10 min to remove the precipitate, and then the absorbance of the supernatant was measured at 532 nm with a microplate reader. The SOD activity was determined according to the WST-1 method. Twenty microlitres of test sample, 20 μl of enzyme diluent and 100 μl of substrate application solution were mixed and incubated at 37 °C for 20 min. The OD values were measured by a microplate reader at 450 nm. The CAT activity was determined according to the visible light method. A 100 μl test sample and working solution were mixed and incubated at 37 °C for 1.0 min, and then termination fluid was added. The OD values were measured by a microplate reader at 405 nm. The GSH-px activity was determined according to the colorimetric method. One hundred microlitres of test sample and 200 μl of GSH were mixed and incubated at 37 °C for 5.0 min, and then working fluid one was added and incubated at 37 °C for 5.0 min. Then, color reaction was performed. The OD values were measured by a microplate reader at 412 nm.
Neuropsychological performance in patients with substance use disorder with and without mood disorders
Published in Nordic Journal of Psychiatry, 2020
Irma Höijer, Tuula Ilonen, Eliisa Löyttyniemi, Raimo K. R. Salokangas
The computerised CogniSpeed tasks [29] were used to measure simple reaction time, automatic and conscious information processing. Simple reaction time subtest of the computerised CogniSpeed test battery performed first. Inhibitory capacity was assessed by the CogniSpeed version of the Stroop Color-Word Test [30]. The test consists of three subtests: (1) Neutral Condition (COL) and (2) Congruous Word Condition CON and (3) Incongruous Word Condition (IN2). COL and CON are related to more automatic and routinized information processing. Incongruous Word Condition (IN2) measures more conscious and effort-intensive processing. Each task begins with a practice session of 10 items and a final session of 50 items. In each subtest, the order of the colors was randomised. The two response buttons were coloured red and blue. When the color and the meaning were incongruent, suppression of word meaning processing was demanded. The Color Reaction times consist of three different conditions, which differ only with regard to the semantic content of the stimuli, neutral, congruous, or incongruous. In every condition, the subjects were asked to respond only to the colour of the letters presented (red or blue).