Supplemental Tests for HIV-1 Infection
Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts in Retroviral Testing, 2020
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.
Optical Imaging Probes
Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman in Molecular Imaging in Oncology, 2008
Exogenous organic dyes in optical imaging of cancer are used mostly for in vitro microscopy because most of the cellular components are optically transparent or nonfluorescent. The use of fluorochromes allows to use longer wavelengths for excitation and emission and to achieve good spectral separation between characteristic emissions of various labeled cellular components. However, in vivo imaging requires fluorochromes with special characteristics: they need to be excitable in a convenient range of wavelengths with corresponding photon energies allowing propagation in the tissues and emitting fluorescence in the range that has minimal absorption and scattering in the body. Recently, nanoparticles and polymers with such fluorescence characteristics were designed and added to armamentarium of in vivo optical imaging. These and other exciting developments are reviewed below.
Routine and Special Techniques in Toxicologic Pathology
Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard in Toxicologic Pathology, 2018
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.
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.
A novel role for an old target: CD45 for breast cancer immunotherapy
Published in OncoImmunology, 2021
Annat Raiter, Oran Zlotnik, Julia Lipovetsky, Shany Mugami, Shira Dar, Ido Lubin, Eran Sharon, Cyrille J. Cohen, Rinat Yerushalmi
Fresh lymphocytes (0.5x106/50 µl PBS) were incubated with the following antibodies for multicolor staining: CD3-PC5.5, CD4-PC7, CD8-KO, CD56-PE, CD16-APC, NKG2D-AF750, CD45RA-APC, CD45RO-PC5.5 and CD14-KO (Beckman Coulter) and with 5 µg/50 µl C24D-FITC peptide for 40 minutes at room temperature. The cells were then washed twice (10 min., 1200 rpm, 4°C). Activated lymphocytes obtained from co-cultures with tumor cells were subjected to the same procedure. To ensure specificity, a Fluorescence Minus One (FMO) control was used to identify and gate cells in the context of data spread due to the presence of multiple fluorochromes in any given panel. In brief, for each sample, two tubes were used: one tube containing cells stained with the antibodies identifying the various PBMC subpopulations and a control peptide-FITC labeled and a second tube containing cells stained with the antibodies identifying the different PBMC subpopulations and with the C24D-FITC peptide. The percentage C24D/CD45 binding on the cell surface in each subpopulation was calculated after subtracting the fluorescence obtained for the control tube. The cells were washed with PBS and suspended in 0.5 ml PBS for FACS (Coulter Navios flow cytometer, Indianapolis, IN, USA). Data were analyzed with the Kaluza software (Beckman Coulter, IN, USA).
IgG4 induces tolerogenic M2-like macrophages and correlates with disease progression in colon cancer
Published in OncoImmunology, 2021
Galateja Jordakieva, Rodolfo Bianchini, Daniel Reichhold, Jakob Piehslinger, Alina Groschopf, Sebastian A. Jensen, Ettore Mearini, Giuseppe Nocentini, Richard Crevenna, Gerhard J. Zlabinger, Sophia N. Karagiannis, Alexander Klaus, Erika Jensen-Jarolim
After 72 h, M2a cells incubated with or without IgG1- or IgG4-ICs were detached using ice-cold PBS/EDTA and washed twice with cell staining buffer (cat.420201 BioLegend, San Diego, CA), for surface marker phenotypization. Then, cells were incubated with a multicolor staining mix of monoclonal antibodies against CD14, CD86, CD11b, CD163, and CD206 or their isotype controls (BioLegend, San Diego, CA) diluted 1:100 in staining buffer for 30 min at 4°C followed by 2x washing with staining buffer. Samples were acquired by FACS Canto II (Becton Dickinson, Franklin Lakes, NJ) and analyzed with the FlowJoTM Software version 10.3 (FlowJo, LLC, Ashland, OR, USA). Several FlowJo plugins were used to improve the Flow cytometer data analyses. First, the FlowAI plugin was used to identify anomalous events and clean the data by removing them. Second, the DownSample plugin was used to reduce the number of events necessary for the representation of all the parameters datasets in a two-dimensional space to 9.000 events. Finally, a machine learning algorithm uniform manifold approximation and projection (UMAP) was used for dimensionality reduction by using the UMAP plugin. For the characterization of each parameter the geometric mean fluorescence intensity (MFI) values that were calculated for each fluorochrome were used. The z‐normalization of MFI for each staining antibody and each donor was performed for M2a, M2a + IgG1, and M2a + IgG4.23 (Supporting information for z-norm calculation).
Related Knowledge Centers
- Antibody
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- Fluorescence
- Macromolecule
- Dye Tracing
- Staining
- Fluorescence Microscope