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Senescent Cells as Drivers of Age-Related Diseases
Published in Shamim I. Ahmad, Aging: Exploring a Complex Phenomenon, 2017
Cielo Mae D. Marquez, Michael C. Velarde
Cell cycle analysis may also be performed using flow cytometry. Flow cytometric analysis uses DNA stains that bind proportionally to the amount of DNA present within the cell [37]. The more dyes taken up, the brighter the fluorescence. This helps to distinguish senescent cells (arrested at G1 or G2/M phases) versus non-senescent cells (quiescent at G0 or undergoing G1/S phase transitions) [27,28]. DNA-binding dyes which are frequently used include propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), 7-aminoactinomycin-D (7-AAD), and Hoechst 33342 [38]. Generally, cells must be fixed and/or permeabilized to allow passage of the dyes, which is otherwise excluded by live cells. Common fixatives applied are alcohol and aldehyde which may be used in combination with a detergent or other permeabilizing reagents. Alcohols fix cells by dehydration and protein denaturation, while aldehydes fix cells by cross-linking proteins and other macromolecules [39].
Flow Cytometry
Published in Wojciech Gorczyca, Atlas of Differential Diagnosis in Neoplastic Hematopathology, 2014
Figure 3.12 illustrates the gating strategy applied in the FC analysis of the lymph node and other solid organs or effusions (spleen is most often analyzed using BM or blood strategy). The nonviable cells are excluded from the analysis by gating on “viability” dye-negative population(s) [7-amino-actinomycin D (7-AAD), propidium iodide (PI), calcein blue, “aqua” viability dye, etc.]. High-grade lymphomas are often characterized by increased numbers of nonviable cells or complete dropout of neoplastic cells, leaving only benign (residual) lymphocytes for analysis. Nonviable cells have tendency for nonspecific adsorption of antibodies leading to nonconclusive or noninterpretable results, and therefore, FC results with increased numbers of nonviable cells have to be interpreted with caution (preferably comparing analysis with and without poorly preserved populations). Some benign and malignant populations may show a “decreased” viability due to nonspecific adsorption of “viability” dyes (e.g., eosinophils; Figure 3.13). Low-grade (small cell) lymphomas tend to remain viable, even when the analysis by FC is delayed.
Structure-Function Elucidation of Flavonoids by Modern Technologies
Published in Dilip Ghosh, Pulok K. Mukherjee, Natural Medicines, 2019
Ritu Varshney, Neeladrisingha Das, Rutusmita Mishra, Partha Roy
The flow cytometer is an important tool having enormous potential for studying various aspects of cells, including cell counting, sorting and biomarker detection. The most important applications of flow cytometry are measurement of cellular DNA content and cell cycle analysis. Proliferating cells undergo five phases of the cell cycle: G0, G1, S, G2 and M. Different phases of the cell cycle have different quantities of DNA content. However, the M phase is indistinguishable from the G2 phase and G0 from G1. Hence, when cell cycle analysis is carried out on the basis of DNA content alone, the cell cycle is commonly described as G0/G1, S and G2/M phases. DNA content or ploidy represents the total number of chromosomes in a cell. Any abnormality (e.g. cancer cells) in the variation in ploidy is observed and this can be detected by flow cytometry. Many modern anticancer drugs act on DNA topoisomerases, which in turn affects DNA replication (Sorenson et al. 1990; Buolamwini 2000; Gamet-Payrastre et al. 2000). These drugs also help in cell cycle arrest and all these phenomena can be confirmed by flow cytometry. The main detection in this system is performed by fluorescence from DNA binding dyes used in the assay. Some important DNA binding dyes usually used for cytometric analysis are propidium iodide, 7-aminoactinomycin-D, Hoechst 33342, 33258 and S769121, TO-PRO-3 and 4′6′-diamidino-2-phenylindole. The advantage of modern flow cytometers is that they can analyse more than a thousand samples per second and, if associated with cell sorters, can then separate and isolate the cells at the same rate.
Evaluation of a docetaxel-cisplatin-fluorouracil-Au complex in human oral carcinoma cell line
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2023
Wannisa Khamaikawin, Kitsakorn Locharoenrat
The test drug was expected to induce the apoptosis of treated cells. The intact plasma membranes of viable cells exclude the 7-aminoactinomycin D (7-AAD) fluorescent dye, whereas the ruptured membranes of late apoptosis/dead cells enable the binding of 7-AAD to double-stranded DNA between base pairs within G-C-rich regions, allowing for the quantification of cell death via flow cytometry [31]. Cell death rates in negative and positive control cells were 0.48 ± 0.03% and 93.20 ± 5.60% (mean ± SD, n = 3), respectively. The rate of cell death almost disappeared between 1 and 2 h of incubation time intervals of the cells treated with the IC50. After 24 h of incubation time with the test drug, late apoptosis cells accumulated (Figure 3). 7-AAD was undetectable in cells treated with Au nanoparticles alone, represented by the FITC channel (KB3). Au nanoparticles were deemed biocompatible as they did not induce an increase in apoptosis. KB cells treated with the docetaxel-cisplatin-fluorouracil-Au complex (KB2) exhibited a significantly higher rate of cell death, at 85.70 ± 1.21% (mean ± SD, n = 3), as compared to those treated with docetaxel-cisplatin-fluorouracil (KB1), at 80.63 ± 0.29% (mean ± SD, n = 3), which could be attributed to the increased availability of docetaxel-cisplatin-fluorouracil in Au nanoparticles. This result was consistent with cytotoxicity analysis, suggesting that the docetaxel-cisplatin-fluorouracil-Au complex tended to enhance the rates of docetaxel-cisplatin-fluorouracil-induced apoptosis.
Schweinfurthin induces ICD without ER stress and caspase activation
Published in OncoImmunology, 2022
Ruoheng Zhang, J.D. Neighbors, T.D. Schell, R.J. Hohl
The second module of the canonical CRT exposure pathway is defined as the apoptotic module. This module involves the activation of caspase 8 and BAX/BAK. Therefore, we investigated if MeSG induces apoptosis in both B16F10 and UACC903 cells. Fluorescent conjugates of annexin V are widely used to identify apoptotic cells because of its high affinity for phospholipid phosphatidylserine (PS).54 In normal healthy cells, PS is located on the inner leaflet of the plasma membrane. However, during apoptosis, PS translocate from the inner to the outer leaflet of the plasma membrane. Therefore, Annexin-V staining cells are considered apoptotic. 7-Aminoactinomycin D (7-AAD)55 is a fluorescent chemical compound with strong affinity for DNA, but it cannot readily pass through intact cell membranes. Hence, cells with compromised membranes (dead cells) will stain with 7-AAD. Generally, Annexin-V (-)/7-AAD (-) cells are considered live cells, Annexin-V (+)/7-AAD (-) cells are at early apoptosis, and Annexin-V (+)/7-AAD (+) cells are at late apoptosis.
Evaluation of cytotoxicity and biodistribution of mesoporous carbon nanotubes (pristine/-OH/-COOH) to HepG2 cells in vitro and healthy mice in vivo
Published in Nanotoxicology, 2022
Yujing Du, Zhipei Chen, M. Irfan Hussain, Ping Yan, Chunli Zhang, Yan Fan, Lei Kang, Rongfu Wang, Jianhua Zhang, Xiaona Ren, Changchun Ge
To detect the association of cell death and ROS, 7-aminoactinomycin D (7-AAD) and DCFH staining were performed via flow cytometry and fluorescence imaging. For flow cytometry, after 48 h exposure mCNTs (10, 100 μg/mL), cells were rinsed with PBS and filtrated by a 40 μm cell filter. Then cells were resuspended with 200 μL PBS, and DCFH-DA probe (10 μM/105 cells) and 7-AAD dye (2 μL/105 cells) were successively added in each tube for 20 min and 10 min. Normal saline-treated group and mechanical damage (freeze-thawing) cells were regarded as control groups. Of note, mechanical damage was occurred in one cycle of freezing and thawing the cells (without mCNTs exposure). All samples were tested immediately by a flow cytometer with 10,000 cells. The division of cross gate was mainly based on blank, only 7-AAD and only DCFH staining control. All of the measurements were repeated for four times. For fluorescence imaging, after the same exposure, cells were washed with PBS, and then 10 μM DCFH-DA probe and 2 μL 7-AAD dye were successively added in each well for 20 min and 10 min. After incubation and washing, the fluorescence microscope (Nikon, Japan) was used to observe the DCFH-DA (green) and 7-AAD (red) signals.