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Progress in Capillary Flow Cytometry
Published in Frances S. Ligler, Jason S. Kim, The Microflow Cytometer, 2019
David King, Amedeo Cappione, Fedor Ilkov, Bruce Goldman, Ray Lefebvre, Rick Pittaro, G. J. Dixon
In this example, Jurkat cells were exposed to a variety of cytoactive compounds (n = 96) with the ultimate goal being the identification of unique cell cycle inhibitors.10,11,12 Following exposure, cells were harvested and then stained with propidium iodide, a cell-permeant DNA-intercalating dye used to measure DNA content.13,14 Relative staining is visualized by flow as red fluorescence and the number of cells in each stage of the cell cycle counted using a histogram as illustrated in Fig. 5.11. An untreated culture has cells present in all 3 phases of the cell cycle while cultures that are blocked at entry to S-phase or M-phase show most cells stuck within the G0/G1 or G2-phases, respectively. Following staining, samples were collected on an EasyCyte then analyzed using the InCyte software package to determine which compounds caused phase-specific blockage. Data interrogation and comparative results are displayed at the high-level experiment level rather than on a single well basis.
Introduction to Biological Light Microscopy
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Jay L. Nadeau, Michael W. Davidson
Another popular traditional probe that is useful in confocal microscopy is the phenanthridine derivative, propidium iodide, first synthesized as an antitrypanosomal agent along with the closely related ethidium bromide. Propidium iodide binds to DNA in a manner similar to the acridines (via intercalation) to produce orange-red fluorescence centered at 617 nm. It also has a high affinity for double-stranded RNA. Propidium iodide has an absorption maximum at 536 nm and can be excited by the 488 or 514 spectral lines of an argon-ion (or krypton–argon) laser, or the 543 line from a green HeNe laser. The dye is often employed as a counterstain to highlight cell nuclei during double or triple labeling of multiple intracellular structures. The structurally similar ethidium bromide, which also binds to DNA by intercalation, produces more background staining and is therefore not as effective as propidium.
Usage of Engineered Virus-Like Particles in Drug Delivery
Published in Jyoti Ranjan Rout, Rout George Kerry, Abinash Dutta, Biotechnological Advances for Microbiology, Molecular Biology, and Nanotechnology, 2022
Sushil Kumar Sahu, Ramakanta Rana, Ashok Kumar Mallik
VLPs are considered as useful biological tools to transfer drugs and desired genetic materials into target cells. Polyoma-VLP is composed of empty capsids and modified polyomavirus DNA (Barr et al., 1979). The DNA uptake happened by an osmotic shock. First, a complex was formed between the empty capsids and the DNA. Then, incubated in the water bath at 37 °C to load the capsids (Ou et al., 2001; Braun et al., 1999). The integrated DNA was protected from DNase degradation (Bertling et al., 1991). This VLP is composed of VP1 and has the potential to incorporate DNA fragments up to 3 kbp (Soeda et al., 1998). Linear, circular, supercoiled, single-stranded DNA, double-stranded DNA, rRNA, and synthetic polymers can be encapsulated into VLPs (Henke et al., 2000). The DNA is complexed with polylysine and is encapsulated in VLPs to protect the DNA against degradation (Soeda et al., 1998). The major structural protein VP1 of the Aleutian disease virus bind to DNA in contrast to minor structural proteins VP2 and VP3 (Will-wand and Kaaden, 1988). It has been reported that VP1 of Simian virus 40 (SV40) binds to the DNA tightly, but the affinity of binding is stronger for single-stranded DNA than for double-stranded DNA (Soussi, 1986). VLPs consisting of VP1 (VP1-VLP) are more efficient in transporting heterologous DNA into host cells or tissues than DNA on its own (Slilaty and Aposhian, 1983). Using VLPs loaded with desired DNA, a long-term and higher level expression was achieved (Krauzewicz et al., 2000). Murine polyomavirus VP1-VLPs showed an enhanced immune response in the mice model. After VP1-VLPs infection, antibodies were accumulated and cells were started to proliferate which may be helpful for vaccination (Clark et al., 2001). VP1-VLPs are shown to be useful tools to incorporate not only DNA but also small biological molecules and to improve the specific delivery of desired molecules to their target cells. Delivery of DNA was experimentally proven by staining DNA with propidium iodide, which intercalates into DNA and was detected under microscopy (Goldmann et al., 2000).
Observation of synergistic antibacterial properties of prodigiosin from Serratia marcescens jx-1 with metal ions in clinical isolates of Staphylococcus aureus
Published in Preparative Biochemistry & Biotechnology, 2022
Yu-Jie Wang, Wei Wang, Zhong-Yu You, Xiao-Xia Liu
Propidium iodide (PI) can pass through the compromised membrane of cells, and it fluoresces upon binding to DNA. Therefore, a fluorescent signal after PI dyeing can indicate membrane damage.[26,27] For examining cell membrane permeability, untreated cells and cells treated with 0.1 μg/mL PG were collected after they were grown for 4 h. The cells, including MSSA (number 19644987) and MRSA (number 19644109), were washed and dissolved in PBS (50 mM, pH 7.4). Cells were dyed using PI and then detected using flow cytometry (FCM, Bectonpikinson, America) using Wang et al.’s protocol.[28] First, 900 μL of cell solution (about 106–107 cells per mL) was stained with 100 μL of PI to achieve a final concentration of 10 μg/mL. The mixture was placed at 25 °C in shade for 15 min and then analyzed using FCM according to the method reported by Suryawanshi et al..[29] About 10,000 cells were analyzed for each sample.
In vitro study on electrospun lecithin-based poly (L-lactic acid) scaffolds and their biocompatibility
Published in Journal of Biomaterials Science, Polymer Edition, 2020
Zhonghua Xu, Peng Liu, Hongyin Li, Mingkui Zhang, Qingyu Wu
Propidium iodide (PI) intercalates into double-stranded nucleic acids and fluoresces. It is excluded by viable cells but could penetrate cell membranes of dead cells. Thus PI staining in this test could be used to show the morphology of BMSCs on different 3D scaffolds after fixed in 4% formaldehyde, not to identify dead cells. The pure and modified PLLA scaffolds seeded with BMSCs after 18 h of incubation at 37 °C with 5% CO2 were washed with PBS × 3 (PH 7.4) and then fixed in 4% formaldehyde for 30 min. After washed with PBS × 3, the fixed scaffolds were immersed in PBS. PI (Santa Cruz) was added to a final concentration of 20 μg/ml in each well, which was incubated and protected from light for 30 min at room temperature. Cells on the scaffolds were then destained in PBS and observed using a fluorescence microscope (Nikon) with 535 nm excitation and 615 nm emission filters.
Spectrophotometric and physicochemical studies on the interaction of a new platinum(IV) complex containing the drug pregabalin with calf thymus DNA
Published in Journal of Coordination Chemistry, 2020
Nahid Shahabadi, Sara Amiri, Hossein Zhaleh
Fixation for all cells in this study was by 4% w/v paraformaldehyde in PBS, pH = 7.4 for 10 min at room temperature. An in situ cell death detection kit (Roche) was used to identify the apoptotic cells by TUNEL (Terminal Uridine deoxynucleotidyl transferase dUTP Nick End Labeling) staining, following the manufacturers protocol. Briefly, all cells were fixed, permeabilized, blocked and incubated with a mixture of fluorescent labeled nucleotides on TDT (terminal deoxynucleotidy transferase) catalyzed by the polymerization of labeled nucleotides to 3/0 H terminals of DNA fragments. The cells were then counterstained with 10 μg/mL of propidium iodide (red) at room temperature for 15 min and washed with PBS. A positive apoptosis control, cells induced into apoptosis by 5% ethanol treatment, were included in each assay. TUNEL positive cells were counted in eight randomly selected fields from each culture under a fluorescent microscope (Olympus AX-70) and apoptotic index was calculated by dividing the number of apoptotic cells by the total cells.