China
Africa
Explore chapters and articles related to this topic
Flow Cytometric Analysis of Human Bone Marrow
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
James G. Bender, Dennis Van Epps
Several flow cytometry methods have been developed using DNA-binding dyes to examine the cycling cells within marrow. The DNA-binding dyes used include PI and Hoechst 33342 (HOE). PI and HOE give similar profiles in identifying cycling cells. The differences are in their excitation emission profiles. As mentioned previously, PI can be excited with a 488 nm laser and emits in the PE channel; therefore, phycoerythrin-labeled probes cannot be used in conjunction with PI. HOE requires UV excitation (350 nm), and therefore requires the use of a second laser to perform immunophenotyping, but permits the use of both FITC and PE-labeled probes. Although HOE is taken up by viable cells, the fluorescence intensity varies considerably between different cell types.78 The problem encountered when combining DNA staining, or measurement of intracellular antigen expression, with surface antigen measurement is the requirement for permeabilization of the cells to permit the entry of the DNA-binding dye or the antibody. A variety of agents have been reported to preserve the surface antigen staining and allow the entry of the DNA-binding dye. These agents include saponin,72 ethanol,68,77 Tween 20,69 and hypotonic conditions.70 An additional important measurement in these types of studies is the determination of the height and width of the fluorescence pulses to detect doublets resulting from agglutination of cells and leading to inaccurate quantitation of the cycling population.79
Basic Science and Molecular Oncology
Published in Manit Arya, Taimur T. Shah, Jas S. Kalsi, Herman S. Fernando, Iqbal S. Shergill, Asif Muneer, Hashim U. Ahmed, MCQs for the FRCS(Urol) and Postgraduate Urology Examinations, 2020
Paul Cleaveland, Vijay Sangar, Noel Clarke
The diagram shows a typical graph from flow cytometry. Flow cytometry allows cells to be sorted depending on DNA content. Cells can be stained with a host of fluorescent markers for various pathways. Hoechst 33342 stains DNA directly. Computer analysis of the fluorescence allows assessment of the amount of DNA in each cell. The graph plots DNA content or fluorescent intensity versus number of cells at the given DNA content. The initial peak shows cell debris. The first peak represents G1 phase and non-proliferating cells. The second peak is G2/M phase, cells that are replicating hence have more DNA within them. The area between these two peaks is S phase, intermediate amounts of DNA.
Detection of Lysosomal Membrane Permeabilization
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Anne-Marie Ellegaard, Line Groth-Pedersen, Marja Jäättelä
Incubate the cells with 5 µg/mL Hoechst-33342 for 2 min for visualization of the nucleus.Other nuclear dyes can be used as well or the nuclear staining can be omitted.
Negatively charged phospholipids doped liposome delivery system for mRNA with high transfection efficiency and low cytotoxicity
Published in Drug Delivery, 2023
Lin Wang, Huanchun Xing, Shuai Guo, Wenbin Cao, Zinan Zhang, Lijuan Huang, Sui Xin, Yuan Luo, Yongan Wang, Jun Yang
The liposomes were prepared by ultrasonic method in the conditions of DOTAP and POPS molar ratio (7:3), total lipid concentration (0.192 mg/mL) and EGFP mRNA concentration (0.027 mg/mL), and then were added to cultured cells in 96-well plates at a volume of 150 μL per well after replacing the new MEM for 0.5, 1, 2, or 4 h. Subsequently, all fluids in each group of wells were removed. The cells were washed twice with phosphate-buffered saline (PBS). Then, fresh MEM was added to the cells to continue cultivation. After 24 h of cell treatment, all liquid in each well was removed, and the cells were washed twice with PBS. Hoechst 33342 was used for nuclear staining. Finally, the cells were observed for the EGFP fluorescence (EX WL 488 nm, EM WL 597 nm) and photographed using an IXM-C high-intensity imaging analysis system.
Legumain/pH dual-responsive lytic peptide–paclitaxel conjugate for synergistic cancer therapy
Published in Drug Delivery, 2022
Shanshan Zheng, Yue Cai, Yulu Hong, Yubei Gong, Licheng Gao, Qingyong Li, Le Li, Xuanrong Sun
To verify cellular uptake of PPP NPs, 2 × 104 cells of MCF-7 cells, HCT116 cells and 4T1 cells were seeded in 24-well plates. One milligram PPP and 0.01 mg coumarin (C6) were dissolved in THF, then the mixture was added dropwise to 1 mL PBS and stirred overnight. Filtered with a 220 nm filter to ensure the unencapsulated C6 was removed, C6-loaded PPP NPs (PPP-C6) were obtained and the C6 concentration was measured using a fluorescent microscopy (Olympus IX73, Shibuya, Japan) with excitation wavelength of 466 nm and emission wavelength of 504 nm. The encapsulation efficiency of C6 was 0.168% which showed the effective loading of PPP-C6. Next, the cells were treated with PPP-C6 at 10 µg/mL for 2 h. The medium was removed and the cells were washed with PBS for three times, fixed with 4% paraformaldehyde for 15 min. Hoechst 33342 was used to stain cell nuclei for 20 min. After washed with PBS for three times, cells were presented by fluorescent microscopy (Olympus IX73, Shibuya, Japan).
MiR-361-5p promotes proliferation and inhibits apoptosis of fibroblast-like synoviocytes via targeting ZBTB10 in rheumatoid arthritis
Published in Autoimmunity, 2022
Aixian Zhang, Rong Lu, Huifang Lang, Min Wu
EdU detection kit (Sangon Biotech, Shanghai, China) was applied to test the proliferation ability of transfected cells. In brief, the EdU solution was first diluted with medium to prepare a 50 μM EdU-containing medium. Then 100 μL EdU-containing medium was injected into the plate and continued treated for 2 h, and rinsed with PBS. Next, we added 150 μL/well aldehyde fixative and fixed the cells for 30 min at 25 °C, and added 150 uL of 2 mg/mL glycine to neutralize the aldehyde. After that, 200 μL of 0.5% Triton-X100 was added to each well for cell permeabilization. Then, incubated the cells with 100 μL Apollo reagent for 30 min in the dark. Finally, 200 μL of 5 μg/mL Hoechst 33342 staining solution was applied to stain DNA. A fluorescence microscope was used to take cell pictures.