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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:

(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.


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