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In Vitro Alternative Methods for the Assessment of Dermal Irritation and Inflammation
Published in David W. Hobson, Dermal and Ocular Toxicology, 2020
David W. Hobson, James A. Blank
The MTT procedures can be performed in tissue culture media containing phenol red and serum. Phenol red, which is present in most tissue culture media, does not interfere with absorbance readings at 570 nm when the pH is maintained below 5.5. This is the purpose for acidification of the isopropanol and SDS-DMF solubilization solutions described below.44 The MTT procedure described below is essentially that of Mosmann.38MTT (Sigma catalog # M2128) is dissolved in phosphate buffered saline (PBS) at 5 mg/ml, filtered through a 0.45 μm filter to sterilize and remove insoluble residue.Following the desired length of cellular exposure to growth factor or toxicant, 10 μl of the 5 mg/ml MTT solution is added per 100 μl of medium. The test plates are then incubated at 37°C for 4 h.100 μl of acidic isopropanol (0.04 N HCl) is added to all wells and mixed thoroughly to dissolve the formazan crystals. Following a 10 to 15 min incubation at room temperature, the OD at 570 nm is determined using a reference wavelength of 630 nm. The plates should be read within 1 h of adding the acidic isopropanol.
Relation Between Contraction and Metabolic Efficiency
Published in Samuel Sideman, Rafael Beyar, Analysis and Simulation of the Cardiac System — Ischemia, 2020
Joseph Kedem, M. Scheinowitz, E. Furman, J. Sonn, H. R. Weiss
Left ventricular utilization of the exogenous substrates glucose, pyruvate, lactate, and free fatty acids was estimated from arterial and coronary sinus blood samples taken under the experimental conditions described above. Free fatty acid concentrations were determined (in triplicate) by the method of Dole,6 as modified by Mosinger13 and Trout et al.19 The Method is based on selective extraction of free fatty acids from the plasma and subsequent titration for the hydrogen ions of the free fatty acids which react with diethyl barbital forming free diethyl barbituric acid. The reaction takes place in a solution of phenol red which changes its color from basic red to acidic yellow. The intensity of the color is determined at 560 nm.
Serological Typing of HLA-A, -B, and -C Antigens
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
An important component of the lymphocytotoxic assay is the source of complement; this is discussed in more detail in the methods in "Complement Testing". The microliter volume ratios of cells/serum/complement is usually 1:1:5, respectively. It is possible to use a 0.5:0.5:2 ratio, but particularly when using fluorescent reading methods, the cells may tend to lie on the sides of the well instead of at the bottom of the well due to the final small volume (3 μl). This will cause difficulty in reading, but such an event can be overcome by spinning the trays in a centrifuge: turn on the centrifuge and as soon as the speed has reached the RPM equivalent of 90 × g, the centrifuge should be switched off without applying the brake. This problem can be avoided by using tissue culture grade plates in which the sera disperse over the bottom of the wells. The disadvantage of these trays is that once the serum has been dispensed into a well, it effectively becomes invisible. The authors suggest the use of phenol red (1 μl of 1 % phenol red per 200 μl of serum) or nontoxic vegetable dyes to stain the sera.
Donor related corneal graft infection: a review of literature and preventive strategies
Published in Seminars in Ophthalmology, 2023
Quality control in eye banking is performed by inspection of donor preservation media for color and turbidity. Phenol red is used as an effective pH indicator used in these media. At nontoxic concentration (0.002%), it effectively provides a clue for contamination. The medium turns colour to yellow with an acidic shift which can occur due to microbial contamination and red on an alkaline change.40 (Figure 1) Optisol GS is a popular intermediate term storage medium that contains the antibiotics gentamicin sulfate and streptomycin sulfate.7 The organ culture medium commonly used in European nations anti-bacterials (streptomycin sulphate 100 mg/mL, penicillin G sulphate 100 mg/mL) and anti-fungals (Amphotericin B with concentration of 0.25 mg/mL) and could store cornea at 37 degree C for about a month. Antibiotics are found to be more effective at warmer temperatures than at hypothermic state as they work better when organisms are metabolically active at warmer temperatures.41 Besides the advantage of storage at room temperature, ease of daily inspection and laboratory testing (BACTEC plus) offers additional advantage of detection of contamination in organ culture.42 Glycerol preservation technique has been adapted by eye banks in India as an adaptation to the COVID-19 pandemic induced scarcity of donor tissues. It is documented that glycerol has antimicrobial properties and the corneal transplantation with glycerol preserved cornea is quite safe for emergency tectonic keratoplasty procedures.43 (Figure 2)
4-Anilinoquinazoline-based benzenesulfonamides as nanomolar inhibitors of carbonic anhydrase isoforms I, II, IX, and XII: design, synthesis, in-vitro, and in-silico biological studies
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Hossam Nada, Ahmed Elkamhawy, Magda H. Abdellattif, Andrea Angeli, Chang Hoon Lee, Claudiu T. Supuran, Kyeong Lee
The activity of the CA-catalyzed CO2 hydration reaction was evaluated employing an applied photophysics stopped-flow instrument. The indicator, Phenol red (at a concentration of 0.2 mM) was added to the CA-catalyzed CO2 hydration reaction after a period of 10–100 s. Phenol red functioned at an absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) and 10 mM NaClO4 (to retain constant ionic strength)35. A CO2 concentration ranging between 1.7 and 17 mM was employed to evaluate the kinetic parameters and inhibition constants33. A minimum of six traces of the starting 5–10% of the reaction were analysed to measure the initial velocity for each inhibitor. In a similar manner, the uncatalyzed rates were evaluated and subtracted from the overall calculated rates36. Stock inhibitor solutions (10 mM) were prepared in distilled–deionized water, and dilutions up to 0.01 nM were prepared with distilled–deionized water after that. Preincubation of the inhibitor enzyme solutions at room temperature to enable the formation of the E-I complex was carried out for 15 min before starting the experiment. The inhibition constants were determined employing PRISM 3, while the kinetic parameters for the uninhibited enzymes were calculated using Lineweaver-Burk plots that comprise the mean of at least three separate measurements with errors ranging to ±5–10% of the stated values37,38.
Preparation and evaluation of oral self-microemulsifying drug delivery system of Chlorophyll
Published in Drug Development and Industrial Pharmacy, 2021
Ling Lin, Sajid Asghar, Lin Huang, Ziyi Hu, Qineng Ping, Zhipeng Chen, Feng Shao, Yanyu Xiao
In the intestinal absorption experiment, the intestine also absorbs water to a certain extent that changes the concentration of the perfusate. The measured results might not reflect the true absorption status, so corrections are needed. At present, the most commonly used method is to add markers (such as: phenol red, 14C-PEG) [48] for calibration. However, markers such as phenol red will interfere with the assay of certain drugs. In the long-term experiment, phenol red might also get partially absorbed. Although 14C is not absorbed, the detection is tedious and the safety is poor. According to comparative studies, the gravimetric method can significantly improve the accuracy of experimental data and significantly reduce the experimental error than the phenol red method. Therefore, in this study, we used the weight correction method to correct the absorption of water [49].