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Supplemental Tests for HIV-1 Infection
Published in Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts, Retroviral Testing, 2020
Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts
Test serum (usually 10 to 20 μl of sample in diluent) is added to each well using a mechanical pipette. Incubation of the slides, usually for 30 min at 37°C, allows specific antibody (if present) to attach to the viral antigens in the infected cells. The slides are then washed in a bath of phosphate buffered saline (PBS) for 15 min with one or two changes of PBS. Following this, the slides must be dried (by air or with a small fan). Conjugate is added in the same manner as the serum. The conjugate is an anti-human immunoglobulin labeled with a fluorochrome (FITC). The fluorochrome is a substance that will fluoresce when exposed to UV light. This occurs when the molecules of the fluorochrome are excited to a higher energy level and emit light of a different wavelength as they return to the ground state. During incubation (usually 30 min, 37°C), the FITC-labeled conjugate will bind to the anti-HIV (if present in the sample). Another wash and drying step follows to remove any unbound conjugate. Buffered glycerol is added to each well (to decrease the refraction of light) and a coverslip is placed over the entire slide. The test should be read immediately, but can be stored in the dark at 4°C and read the following day; alternatively, the coverslip can be sealed with nail polish and the slides stored refrigerated for days before reading. The slides are examined under UV light with the aid of a fluorescence microscope.
Bacteriocins as Anticancer Peptides: A Biophysical Approach
Published in Ananda M. Chakrabarty, Arsénio M. Fialho, Microbial Infections and Cancer Therapy, 2019
Filipa D. Oliveira, Miguel A.R.B. Castanho, Diana Gaspar
Flow cytometry is based on the movement of cells or micrometric particles through a channel subjected to a laser beam [109]. This technique measures the light absorbed, scattered, or emitted by a single particle or cell as a result of its physical properties [109], providing information on cell size, granularity, intracellular complexity, and protein composition [110]. In flow cytometry technique cells are usually stained with fluorescent dyes [109]. The evolution of flow cytometry associated with the use of new fluorochromes or probes emphasized this technique’s applicability in the evaluation of cell viability, membrane structure integrity, and membrane potential in individualized cells [109]. In fact, the use of fluorescence probes in flow cytometry allows us to measure the fluorescence intensity as a result of the metabolic activity of the cells [17]. Overall, the probes used in flow cytometry can be organized into nucleic acid binding and metabolic, cellular, or protein binding probes [109]. Since flow cytometry allows its users to assess different parameters, such as membrane integrity and potential, at a single-cell level it may represent a more accurate method to report on the cells’ function than the traditional cell viability– or cell growth–based methods [109].
Routine and Special Techniques in Toxicologic Pathology
Published in Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard, Toxicologic Pathology, 2018
Daniel J. Patrick, Matthew L. Renninger, Peter C. Mann
The term “fluorescence” refers to the property of some substances (fluorophores) to absorb light of a certain wavelength called excitation light and simultaneously reemit it at a longer wavelength referred to as emission light. The difference in wavelength between excitation and emission is known as Stokes’ shift and is fundamental to fluorescence labeling (Lichtman and Conchello 2005). Some substances (e.g., vitamin A and porphyrins) fluoresce naturally under ultraviolet excitation, which is primary or autofluorescence. Structures within tissues can also be made to fluoresce by the addition of a fluorochrome, termed secondary fluorescence. Each fluorochrome will fluoresce under light within a range of wavelengths, but optimal fluorescence occurs at a particular wavelength called the excitation peak. Fluorochrome labeling can identify cells, subcellular components, and other materials with a high degree of specificity and a high degree of sensitivity since only an extremely small number of fluorescent molecules are needed for detection by the human eye or digital sensor.
Left ventricular assist device implantation causes platelet dysfunction and proinflammatory platelet-neutrophil interaction
Published in Platelets, 2022
Tiago Granja, Harry Magunia, Patricia Schüssel, Claudius Fischer, Thomas Prüfer, David Schibilsky, Lina Serna-Higuita, Hans Peter Wendel, Christian Schlensak, Helene Häberle, Peter Rosenberger, Andreas Straub
Samples were measured on a FACSCanto II flow cytometer (BD) equipped with three air-cooled laser lines: a 405 nm laser as solid-state diode in violet, a HeNe 633 nm red laser, and a solid-state 488 nm blue laser. Each measurement was performed in duplicates. BD FACSDiva software (Version 6, BD) was used for calibration and acquisition procedures. The flow cytometer was calibrated routinely using cytometer setup and tracking beads as recommended by the manufacturer. A compensation protocol was set up accordingly by plotting all colors against each other within the same laser line and also across different lasers. For compensation of potentially upregulated activation markers such as CD62P and CD11b blood samples were stimulated for 30 min at 37°C with 1 U/ml Thrombin (Chrono-Par P/N 386, Chrono-log, Havertown, PA, USA) , or 20 µM ADP (Mölab, Langenfeld, Germany), or 500 ng/mL LPS (Sigma, Taufkirchen Germany) and stained with single fluorescence-labeled antibodies against single epitopes to set up maximum fluorescences compared to untreated controls. A fluorochrome matrix with less than 20% overlap between respective colors was defined. The compensation matrixes were readjusted using FlowJoTM software (Version 10, BD) and applied to all acquisitions.
Microsurgical varicocelectomy effect on sperm telomere length, DNA fragmentation and seminal parameters
Published in Human Fertility, 2022
Sandra Lara-Cerrillo, Josep Gual-Frau, Jordi Benet, Carlos Abad, Juan Prats, María José Amengual, Jordi Ribas-Maynou, Agustín García-Peiró
The following method was developed to obtain a relationship between fluorescence intensity and number of fluorochrome molecules in order to extrapolate results to absolute TL. Briefly, fluorescence analysis of a mix of spheres with different numbers of FITC fluorochromes attached (Sphero Calibration Particles Rainbow - RCPs; 8 peaks, 3.0–3.4µm, Spherotech; IL, USA) was performed using the same image-capturing conditions as qFISH-PNA. From the eight spheres groups of the kit, three were in the same fluorescence intensity range as sperm telomeres. These values were used to obtain a linear equation relating arbitrary units of fluorescence intensity to the number of FITC-fluorochromes attached to the spheres described on the datasheet. Knowing that PNA probes for telomere labelling are 18bp long and attached to just one FITC molecule, the former equation was converted to relate fluorescence intensity and absolute TL. This equation was validated using the 1301 cell-line, derived from a human T-cell leukaemia (telomeres ∼70 kb) (Wang et al., 2013) (Sigma-Aldrich; USA) and sperm from 10 normozoospermic donor samples (telomeres 10–20kb) (Thilagavathi et al., 2013).
Susceptibility to the acute toxicity of acrylonitrile in streptozotocin-induced diabetic rats: protective effect of phenethyl isothiocyanate, a phytochemical CYP2E1 inhibitor
Published in Drug and Chemical Toxicology, 2021
Fang Li, Ying Dong, Rongzhu Lu, Bobo Yang, Suhua Wang, Guangwei Xing, Yuanyue Jiang
The production of intracellular ROS was analyzed using a detection kit according to the manufacturer’s instruction. DCFH-DA is oxidized into fluorescent DCF by ROS in tissue. The oxidation of this molecule to the fluorochrome DCF results in green fluorescence. Briefly, brain tissues were homogenized in phosphate buffer (0.1 mol/L, pH 7.4) and then were centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was incubated with 10 µL of DCFH-DA (1 mmol/L) and then stained in darkness at 37 °C for 30 min. The fluorescence was read on a SynergyH4 microplate spectrophotometer (BioTek, Winooski, VT) at 495 nm excitation and 529 nm emission wavelengths. The intensity of this fluorescence is generally considered to reflect the ROS levels. The results were expressed as DCF-fluorescence/mg protein.