Cells in the logarithmic growth phase were digested with 0.25% trypsin, dispersed into single cells, and suspended in DMEM containing 10% FBS. The cells were then inoculated into dishes containing 10 ml of medium at 37°C to a density of 50 cells per dish. The dishes were then rotated gently to evenly distribute the cells. After incubation for 24 h, the supernatant was discarded, and the cells were carefully washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and stained Giemsa stain (Beyotime Biotechnology, Shanghai, China) for 30 min. The dye solution was then slowly removed with running water, and the cells were air dried. Finally, the dish was inverted and overlaid with transparent film with a grid, after which the numbers of clones were counted under a microscope.
Giemsa stain
Manufactured by Beyotime
Sourced in China
Giemsa stain is a laboratory reagent used for staining blood smears and other cytological specimens. It is a polychromatic stain that provides differential coloration of cellular components, allowing for the identification and differentiation of various types of cells, including red blood cells, white blood cells, and platelets. The Giemsa stain is widely used in clinical diagnostics, hematology, and research applications.
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5 protocols using giemsa stain
1
Clonogenic Assay for Cell Viability
2
Colony Formation Assay for PWP1
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At 48 h post-transfection with PWP1 plasmids or shPWP1, cells were seeded in three 6-cm cell culture dishes (1000 cells/dish) and incubated for 12 days in RPMI-1640 medium containing 10% fetal bovine serum (FBS). The plates were then washed with PBS and incubated with Giemsa stain (Beyotime Biotechnology, Shanghai, China) before counting the number of colonies consisting of >50 cells.
3
Metaphase Chromosome Analysis of MIR503HG Knockout hES-H9 Cells
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The MIR503HG knockout hES‐H9 cells were cultured until reaching 80% confluency. Colchicine (ST1173, Beyotime) was added to the medium and incubated for 3 h at 37°C, and then cells were digested into single cell by Accutase (07920, STEMCELL Technologies). The cells were resuspended in 75 mm KCl solution for 30 min and then incubated in a fixative containing ethanol/acetic acid (3:1, v/v) for 20 min at RT. 20 µl cells suspension were applied to each clean slide. Dyed by Giemsa stain (C0131, Beyotime) for 30 min at RT, 20 metaphases were counted for each sample, and then chromosome analysis were performed using the Leica DMRB epifluorescence microscope.
4
Vaginal Smear Method for Rat Estrous Cycle Identification
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Vaginal smear method was used to identify the estrous cycle of rats. A small amount of normal saline was sucked by a pipette into the rats’ vagina, and mechanically dissociated several times. The vaginal fluid was sucked out and air-dried. The air-dried smear was stained with Giemsa stain (C0133, Beyotime Biotechnology Co., Shanghai, China), punched, dried, and observed under a microscope.
As shown in FigureS1 , during proestrus, there were large number of round nucleated epithelial cells, a few white blood cells, and a few anucleated keratinized epithelial cells. During estrus, there appeared almost all large, flat, non-nucleated keratinocytes. In the diestrus, large number of keratinized epithelial cells, and a small number of white blood cells and nucleated epithelial cells gathered in a pile. During the metestrus, there existed a large number of white blood cells, a small number of round nucleated epithelial cells. The red arrows represent nucleated epithelial cells, the green arrows represent keratinized squamous epithelial cells, and the black arrows represent white blood cells.
As shown in Figure
5
Fibronectin-Mediated Cell Adhesion Assay
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Fibronectin (Sigma, MO, USA) was coated onto a 24-well plate. MV4-11, Kasumi-1, and U937 cells (1 × 10 6 cells per well) were seeded into the 24-well plate. Subsequently, the cells were allowed to adhere for 1 h at 37 °C. Unbound cells were gently washed away with PBS, and the bound cells were fixed with 4% paraformaldehyde solution. Then, the cells were stained with Giemsa stain (Beyotime, China) for 30 minutes, followed by gentle washing with PBS 1 to 2 times. Finally, the cells were placed under a microscope for imaging.
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