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HLA-DR and -DQ Serotyping
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
For cell preparations in which the B cells have been labeled with FITC-anti-immunoglobulin, ethidium bromide solution is added. Ethidium bromide is carcinogenic and appropriate precautions should be taken when handling powder and solutions. The stock solution consists of ethidium bromide, 100 μg/ml in PBS. Make 100 ml of stock solution; this should be kept in the dark at 4°C. To prepare the working solution, take 0.3 ml of stock and add 20 ml of PBS/5% EDTA. The working solution can be aliquoted into 1 or 0.5 ml amounts and kept frozen until needed. Add 0.5 μl of solution to each well. The plates are read using an inverted epifluorescence microscope in a darkened room. With this method it is possible to estimate both B and T cell death. Under fluorescence, live B cells have a green cap and dead B cells are red with a green cap; live T cells are only visible if the plate is viewed with normal light and dead T cells are red without a cap.
Practical Approach to Molecular Biology in Hematopathology
Published in Harold R. Schumacher, William A. Rock, Sanford A. Stass, Handbook of Hematologic Pathology, 2019
Anwar Mikhael, Harold R. Schumacher
PCR requires knowledge of the DNA sequences of interest. Two sequence-specific primers can be prepared synthetically to hybridize to the DNA sequences of interest. The two sites should not be separated by a large stretch of DNA; rather, it is best utilized with DNA segments less than 1 kb. In the event that the primers are separated by large DNA segments, the use of c-DNA, generated by reverse transcription of m-RNA, as a template, overcomes this problem. Repeated cycles of denaturation, annealing, and extension allow the rapid amplification of the DNA segment of interest. The amplified DNA can be visualized on an electrophoretic DNA gel stained with ethidium bromide (Fig. 3).
Structure-Activity Relationships
Published in Danilo D. Lasic, LIPOSOMES in GENE DELIVERY, 2019
A cryo-EM of a similar complex is shown in Figure 9-2A. This is DNA complexed to commercially available DOTAP liposomes. We could not observe DNA fibers. In contrast to these complexes which were shown to be inactive upon systemic administration (Lasic et al., unpublished), we have studied several complexes which were shown to transfect in vivo as measured by expression of CAT protein in the mouse lungs 1 day after tail vein injection. A strikingly different picture was seen. Instead of loose, large, fragile aggregates, smaller dense and compact colloidal particles could be observed (see Figure 7-10A). In some samples locally ordered structures could be seen (Figure 9-2B). Noting two periodicities, a longer one about 5 to 7 nm and a shorter one in the range 3 to 4 nm, we performed small-angle X-ray scattering (SAXS) studies which reproduced EM-determined periodicities completely. As controls, naked DNA, unreacted liposomes, as well as “in vivo-inactive” complexes, as shown in Figure 9-2A, did not yield any reflections. Simple tabletop centrifugation experiments have shown that the active complexes, containing no visible flocculae can be easily (partially) pelleted, indicating that active complexes contain dense particles and sedimentation coefficients over 500 Sv for that fraction of the sample were calculated from Equation 7-1. Ethidium bromide fluorescence studies also showed that in these complexes DNA was inaccessible for the fluorescent dye intercalation indicating good DNA protection.
Combination of PAKs inhibitors IPA-3 and PF-3758309 effectively suppresses colon carcinoma cell growth by perturbing DNA damage response
Published in International Journal of Radiation Biology, 2023
Muzaffer Dukel, Kayahan Fiskin
To detect DNA damage in colon cancer cells, we performed the alkaline Comet assay as previously described by Singh and colleagues (Singh et al. 1988). Briefly, after 48 h of drug treatment of IPA-3 (15 μM), PF-3758309 (15 μM), and in pair at 4:2 μM, SW620 and Colo 205 were harvested and then mixed with low melting point agarose at a ratio of 0.75%, and then they were covered by a coverslip. The slides were then incubated in the lysis solution at +4 °C. Cells were treated with an alkali solution to unwind and denature DNA. Slides were transferred to the electrophoresis chamber from alkali solution and samples were run for 25 min at 1 V/cm. Next, cells were stained with 75 ml of 20 mg/ml of ethidium bromide solution at room temperature for 15 min. Slides were imaged by an Olympus fluorescence microscope.
Effect of bacterial toxin identified from the Bacillus subtilis against the Cnaphalocrocis medinalis Guenée (Lepidoptera: Crambidae)
Published in Toxin Reviews, 2023
Ramakrishnan Ramasubramanian, Sengodan Karthi, Sengottayan Senthil-Nathan, Haridoss Sivanesh, Narayanan Shyam Sundar, Vethamonickam Stanley-Raja, Govindaraju Ramkumar, Kanagaraj Muthu-Pandian Chanthini, Prabhakaran Vasantha-Srinivasan, Khaloud Mohammed Alarjani, Mohamed S Elshikh, Ahmed Abdel-Megeed, Patcharin Krutmuang
Using the standard (Sambrook et al. 1989) protocol, the total genomic DNA was extracted. Isolated DNAs were dissolved in 50µl of TE buffer (10mM Tris–HCl, 1mM EDTA, pH 8.0) and stored at 20° C until used. We used the universal bacterial forward primer 27F (5′- 168 AGAGTTTGATCATGGCTCAG-3′) and the reverse primer 1492R (5′- TACGGCTACCTTGTTACGACTT-3′) to amplify the 16S rRNA gene.PCR amplification was performed at 94°C for 5min, followed by 35 cycles at 94°C for 1min, 50°C for 1min, 72°C for 2min, and a final extension at 72°C for 20min. In order to determine the size of the amplified products, electrophoresis on a 1% agarose gel was conducted. Staining ethidium bromide was then performed under UV light. The sequences were submitted to the GenBank database under the accession number MW365355.
Identification of the first-in-class dual inhibitors of human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase 1 (IDO 1) with strong anticancer properties
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Barbara Kaproń, Anita Płazińska, Wojciech Płaziński, Tomasz Plech
Determination of the inhibitory effect of compounds 1-10 against human topoisomerase Iiα was performed using Human Topoisomerase II Relaxation Assay (Inspiralis Ltd, Norwich, UK) and Topoisomerase II Drug Screening Kit (TopoGEN Inc., Buena Vista, CO, USA) following the manufacturer’s instructions. Briefly, the increased concentrations of the compounds were mixed with the reaction mixture, incubated according to the protocol and, after reaction termination, the samples were analysed by electrophoresis using 1% agarose gel containing 0.5 mg/mL of ethidium bromide in Tris-Acetate-EDTA (TAE) buffer. Visualisation of the bands was performed using an enhanced chemiluminescence system (Syngene G:BOX Chemi XT4, Cambridge, UK). For the quantitative determination of topoisomerase II inhibition, images were densitometrically scanned.