CGH Protocols （一）

 

Metaphase chromosome preparation

Materials:
RPMI 1640 medium
fetal calf serum (FCS), 20%
Colcemid (e.g. Boehringer Mannheim cell biology reagents, Best.-Nr. 295892)
cell cuture flask
Phythemaglutinin, PHA-L (Seromed, M 5030)
CO₂ cell culture incubator
50 ml Nunc/Falcon tubes
15 ml Nunc/Falcon tubes
KCl (0.075 M, 0.055% ?)
Fixative (methanol/acetic acid 3 : 1)
glass microscopy slides

Amounts per 5 ml blood:
40 ml RPMI 1640 Medium
10 ml FCS (20%)
5 ml peripheral blood (anticoagulation by heparin)
1.5 ml PHA
1 cell culture flask, e.g. Falcon 250 ml
prepare up to 10 flask (1 flask will yield about 50 slides)

Steps:

1. Incubate culture for 72 hours in CO₂ cell culture incubator, mix flask 1-2 times per day
2. Add Colcemid (about 45 min before harvesting)
3. Make 2 aliquots and transfer cell into 50 ml Falcon tubes
4. Incubate in cell cuture incubator or 37°C water bath for additional 45 min
5. Centrifuge for 10 min at 1000 rpm
6. Remove supernatant e.g. with a cell culture pipettor until 5 ml remain
7. Gently add 40 ml KCl (0.075 M, 37°C), first 5 ml drop by drop (hypotonic treatment)
8. Incubate for 25 min in 37°C water bath
9. Centrifuge 10 min at 1000 rpm
10. remove supernatant, leave about 5 ml, resuspend pellet
11. Add 2 ml fixative, mix well
12. Add fixative until 40 ml, mix meanwhile
13. Repeat steps 9 - 12 until the pellet is white (at least 4 times)
14. After removal and resuspension of the pellet, transfer cell in 15 ml Falcon tube
15. Repeat steps 9 - 12, add just 10 ml fixative
16. Remove fixative until about 2 ml final volume
17. Resuspend pellet and apply suspension on slides:

• Cool slides to -20°C (e.g. put about 10 slides in a cuvette in the -20°C freezer and keep the cuvette on ice while preparing the metaphase slides)

• take one slide and moisten it by breathing from very close

• either drop 50-100 µl of the suspension on the slide or apply the same volume to the inclined slide (the fast draining and drying of the fluid is usually an indication for good spreading)

• let the susension begin to dry (the fluid film starts to retract)
• put the slide briefly in 70% acetic acid

(the acetic acid step is a washing step in particular for remowing the cytoplasm, in addition it may help for the spreading of the chromosome; the cell membranes attach to the surface of the glass slides and are disrupted by the liquid flow; the temperature difference between the cell and the glass slides may help in the disruption of the cell membranes. If the weather conditions are favorable the 70% acetic acid washing step may be omitted; in our experience the best metaphases spreads occur on dry and sunny days.

18. Air dry the chromosome slide, check for chromosome spreading and cytoplasm debris in a phase contrast lab microscope, adjust volume of fixative so that the density of nuclei/metaphases is appropriate

19. If the conditons are favorable, prepare a batch of metaphases spreads

20. Keep slide in a box at room temperature (up to about 1-2 months); metaphase spreads may be kept longer at -80°C or in 70% ethanol at 4°C.

21. Keep fixative with lymphcates at -20°C until the preparation of new slides. Add new fixative and wash cell before the preparation of new metaphase spreads.