Cells blocked in mid-division were fixed, swollen in hypotonic solution, dropped on slides, stained, and observed [ Rothfels and Siminovitch, 1958; Rooney and Czepulkowski, 1986].
Operation method
Program 16.7 Chromosome Preparation
Principle
Cells blocked in mid-division were fixed, swollen in hypotonic solution, dropped on slides, stained, and observed [ Rothfels and Siminovitch, 1958; Rooney and Czepulkowski, 1986].
Materials and Instruments
D-PBS 0.25% trypsin Cultured cells in log phase Colchicine amide Move 1. 2×104~5×104 cells/ml ( 4×103~1×104cells/cm2 ) 20 ml were incubated in 75 cm2 flasks. Caveat The staining solution must be poured off because oxidized staining solution scum will remain on the slide. Even when staining in a flat dish, the staining solution must not be poured out, but must be replaced with water from the bottom up. For more product details, please visit Aladdin Scientific website.
Hypotonic solution Methanol acetate fixative Giemsa stain DPX or Permount sealer Ice
Centrifuge tubes Pasteur pipettes Slides Coverslips Slide cassettes Low speed centrifuge Vortex mixer
2. When the cells are in the logarithmic growth phase after about 3.5 days, add 0.2 ml of 1×10-5 mol/L colchicine amide (prepared with D-PBSA) to the existing medium in the culture flask at a final concentration of 1×10-7 mol/L.
3. After 4-6 hours, gently remove the medium, add 5 ml of 0.25% trypsin and incubate for 10 min.
4. Centrifuge the cells in trypsin solution and discard the supernatant trypsin.
5. Resuspend the cells with 5 ml of hypotonic solution (0.04 mol/L KCl; 0.025 mol/L sodium citrate), and leave for 20 min at 37℃.
6. Add an equal amount of freshly prepared ice-cold methanol acetate (one part glacial acetic acid to three parts anhydrous methanol or ethanol, freshly prepared and kept on ice), mix constantly, and centrifuge at 100 g for 2 min.
7. Discard the supernatant mixture, shake the cell pellet on a vortex mixer, and slowly add freshly prepared methanol acetate while mixing.
8. Cells were allowed to stand on ice for 10 min.
9. Centrifuge the cells at 100 g for 2 min.
10. Discard the supernatant methanol acetate and resuspend the cell pellet with 0.2 ml of methanol acetate solution by shaking until the cells are evenly dispersed.
11. Pipette a drop of the cell suspension onto the tip of the pipette and drop it onto a cooled slide from approximately 12 inches (30 cm), tilting the slide to allow the droplet to flow down and spread out.
12. Dry the slide quickly over a beaker of boiling water and observe it with a phase contrast microscope. If the cells spread evenly without touching each other, prepare more slices with the same concentration of cells. If the cells appear in overlapping piles, prepare another drop slide after diluting the suspension 2 to 4 times. If satisfactory, prepare more slices, if not, repeat the procedure for further dilutions.
13. Perform Giemsa cell staining:
(a) Place the slides on a rack which rests on the sink.
(b) Completely cover the slide with a few drops of pure Giemsa stain and stain for 2 min.
(c) Flood the slide with about 10 times the volume of water.
(d) Leave for another 2 min.
(e) Rinse off the diluted stain with running water.
(f) Finally, rinse the slides one by one with water to remove the dye precipitate.
(g) Observe the staining under the microscope, and if satisfactory, dry the slide thoroughly and seal it with a #00 coverslip and DPX or Pennoum. 