Protocol for Chromosome Preparation in Human Cancer Cells
GUIDELINE
Corresponding cell lines have now been established for most cancers. Cancer cells cultured in vitro mostly belong to the continuous cycle cells, which can be used to prepare chromosomes. The conventional method for preparing animal and human chromosomes at home and abroad is the air-drying method.
METHODS
- Vigorous growing cells are collected and blown to disperse. Pretreat by adding colchicine to reach a final concentration of 1 μg/ml.
- After 6-8 hours of pretreatment, the cells are centrifuged at 800 rpm for 7 min and the supernatant is removed.
- The cells are resuspended by adding 1 ml of 0.075M KCl solution and blowing gently. Then makeup 7 ml of KCl solution and hypotonic for 20 min at room temperature.
- Add 3-4 drops of Carnoy fixative and centrifuge at 800 rpm for 7 min to remove supernatant.
- Add 3 ml of fixative slowly along the wall of the tube. After 2 min, blow gently with a pipette to disperse the cells. After fixation for 10 min, centrifuge to remove supernatant.
- Add 3 ml of newly prepared fixative and blow well, fix for 10 min, and centrifuge at 800 rpm to remove supernatant. Repeat the procedure.
- After removing the supernatant, 0.2-0.5 ml of fixative is left, and it is blown with a pipette to make a cell suspension.
- Pipeted a drop of cell suspension on an ice-cold slide (soaked in 80% ethanol and refrigerated at 0-4°C), blowing it away and overheating it for about 5 s.
- Blow-dried by a fan. Place in a staining vat with Giemsa staining solution, stain for 20 min, wash with water, blow-dry, and examine microscopically.
Creative Bioarray Relevant Recommendations
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NOTES
A method of preparing chromosomes as opposed to the sectioning and pressing methods used in the past. The cell suspension is first prepared, and then the suspension is dropped on a slide so that the cells and chromosomes are dispersed and adhere to the slide, which is dried naturally or by a blower and is ready for staining and examination.
