In Vitro Alternative Methods for the Assessment of Dermal Irritation and Inflammation
David W. Hobson in Dermal and Ocular Toxicology, 2020
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.
Culture systems for the human embryo
David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham in Textbook of Assisted Reproductive Techniques, 2017
When discussing pH, it is worth considering that the pHi of the embryo is around 7.2 (136–138), while the pH of the media routinely range from 7.25 to 7.4. Specific media components, such as lactic and amino acids, directly affect and buffer pHi, respectively. Of the two isomers of lactate, Dand L-lactate, only the L-lactate form is biologically active. However, both the Dand L-lactate forms decrease pHi of the embryo (138). Therefore, it is advisable to use only the L-isomer of lactate and not a medium containing both the Dand L-lactate forms. While high concentrations of lactate in the culture medium can drive pHi down (138), amino acids increase the intracellular buffering capacity and help maintain the pHi at around 7.2 (64). As the embryo has to maintain pHi against a gradient when incubated at pH 7.4, it would seem prudent to culture embryos at a lower external pH. The pH of a CO2/bicarbonate-buffered medium is not easy to quantitate. A pH electrode can be used, but one must be quick, and the same technician must take all readings to ensure consistency. A preferred and more accurate approach is to take samples of medium and measure the pH with a blood-gas analyzer. A final method necessitates the presence of phenol red in the culture medium and the use of Sorenson’s phosphate buffer standards. This method allows visual inspection of a medium’s pH with a tube in the incubator and is accurate to 0.2 pH units (20, 46).
Staphylococcus aureus
Peter M. Lydyard, Michael F. Cole, John Holton, William L. Irving, Nino Porakishvili, Pradhib Venkatesan, Katherine N. Ward in Case Studies in Infectious Disease, 2010
Culture: S. aureus and other staphylococci grow well on blood agar after 24–48 hours of incubation at 37°C. Staphylococci are facultative anaerobes. Colonies of S. aureus are large, smooth, glossy domes with an entire edge. If held at room temperature the colonies will change color from white to a golden yellow (ergo – S. aureus, the golden staphylococcus). Almost all strains of S. aureus are β-hemolytic due to their secretion of cytotoxins (Figure 9). Mannitol-salt agar containing 7.5% sodium chloride and mannitol as the carbon source is a useful selective and differential medium to recover staphylococci from clinical specimens, for example skin, that contain a mixed microbiota. Most bacteria other than staphylococci are inhibited by this concentration of NaCl and S. aureus can ferment mannitol whereas most other staphylococcal species cannot. Incorporation of the pH indicator, phenol red, reveals colonies fermenting mannitol because acid produced by the colony changes the color of the agar from pink to yellow (Figure 10).
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)
Coumarins inhibit η-class carbonic anhydrase from Plasmodium falciparum
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Simone Giovannuzzi, Viviana De Luca, Alessio Nocentini, Clemente Capasso, Claudiu T. Supuran
The CA-catalysed CO2 hydration activity has been measured with an Applied Photophysics stopped-flow instrument17. The used pH indicator was phenol red (at a concentration of 0.2 mM), working at the absorbance maximum of 557 nm. 10 mM HEPES (pH 7.4) was employed as a buffer, in the presence of 10 mM NaClO4 to maintain the ionic strength constant. The initial rates of the CA-catalysed CO2 hydration reaction were followed up for a period of 10–100 s. The substrate CO2 concentrations ranged from 1.7 to 17 mM for determining the inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used to determine the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionized water with maximum 5% DMSO, and dilutions up to 10 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 1–6 h prior to the assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using Prism 3 and the Cheng-Prusoff equation, as reported previously18–20, and represent the mean from at least three different determinations. The PfaCA concentration in the assay system was 12.38 nM. The human/protozoan enzymes were recombinant proteins obtained in-house, as described earlier12,16.
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.
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