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Experimental procedure
1. Culture Preparation
1) Thaw the bone marrow cell chromosome synchronization kit culture tube or flask at room temperature or 37 ± 2 ℃.
2) Under sterile conditions, add 1ml of bone marrow (the amount added depends on the sample cell density) to a test tube or dispense 5ml bottled medium into a cell culture flask and add the sample.
3) Cover the culture tube.
4) Mix the material in the culture tube (or culture bottle), place it horizontally (on its flat surface), and incubate at a temperature of 37 ± 2 ℃. There is no need to add CO2, because the composition of the medium is designed to shut down the optimal lymphocyte proliferation in the system.
2. Training synchronization
1) After 48-72 hours of incubation, add 0.1 ml of solution A and incubate overnight.
2) After incubation, add 0.1 ml of solution B and incubate for 5 hours (no need to wash cells).
3) Add 20ul colchicine (10ug / ml) and incubate at 37 ± 2 ℃ for 60 minutes (approximately). If you want to obtain a greater number of early and mid-phase split phases, you can reduce the incubation time to 15 minutes.
3. Chromosome fixation and slide preparation
1) After incubation, if necessary, transfer the culture to a test tube and separate it with a centrifuge for 3-4 minutes at a speed of 2.000 per minute (faster speed will not damage cells)
2) Discard the supernatant, taking care not to loosen the cell particles (it is recommended to leave approximately 0.5ml of material in the test tube).
3) Resuspend the particles forcefully.
4) Add 5ml of hypotonic solution (dropwise into H2O, 0.56% potassium chloride) and mix well.
5) Incubate at room temperature for at least 7 minutes.
6) Separate with a centrifuge for 3-4 minutes at a speed of 2.000 per minute.
7) Discard the supernatant according to step 2.
8) Resuspend the particles forcefully.
9) Add 5ml of Ibraimov solution (dropwise into H2O, 5% acetic acid) and mix well.
10) Separate immediately with a centrifuge for 3-4 minutes at a speed of 2.000 per minute.
11) Discard the supernatant according to step 2).
12) Resuspend the particles forcefully.
13) Add 5ml of fixative (methanol: acetic acid, 3: 1) and mix well.
14) Repeat steps 10-13.
15) Immediately separate with a centrifuge for 3-4 minutes at a speed of 2000 per minute.
16) Carefully discard almost all of the supernatant.
17) Add a few drops of freshly prepared fixative, taking into account that the number of drops should be suitable for the cell concentration in the particles.
18) Drop a small drop of cell suspension onto a clean and wet microscope slide.
19) Put into Optichrome chromosome disperser and evaporate under constant conditions of correct temperature and relative humidity.
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