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℃ 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℃), first 5 ml ｄｒｏｐ by ｄｒｏｐ (hypotonic treatment)

8.Incubate for 25 min in 37℃ water bath

9.Centrifuge 10 min at 1000 rpm