Lymph Node Method: 
1.Place lymph node into 2-3ml of RPMI 1640 20% FBS with Penicillin/ 
streptomycin/fungazone in a sterile petri dish. Mince the lymph node 
very finely using a scalpel blade until it become a milky consistency. 

2. Set up 3 cultures as follows (Note: set-up more if sample size 
allows) 
Culture 1: Place cell suspension into 10ml of RPMI 20%FBS in a 25cm 
flask such that the final viable cell concentration is 1000 000/ml. 
Incubate at 37C for 24 hours, adding 0.5ml of colcemid 1ug/ml (final 
concentration 0.05ug/ml) for 60 minutes. Harvest as usual. 
Culture 2: Place cell suspension into 10ml of RPMI 20%FBS in a 25cm 
flask such that the final viable cell concentration is 1000 000/ml. 
Incubate at 37C for 48 hours, adding 0.5ml of colcemid 1ug/ml (final 
concentration 0.05ug/ml) for 60 minutes. Harvest as usual. 
Culture 3: Place cell suspension into 10ml of RPMI 20%FBS in a 25cm 
flask such that the final viable cell concentration is 1000 000/ml. 
Incubate at 37C for 72 hours, adding 0.5ml of colcemid 1ug/ml (final 
concentration 0.05ug/ml) for 60 minutes. Harvest as usual. 
Note: if chromosomes appear too short decrease exposure to colcemid to 
45 minutes. 

3.Harvest: 
a.Transfer culture to harvest tubes and centrifuge for 10 minutes at 
1000rpm 
b.Discard supernatent 
c.Mix cell pellet in 5ml KCL and incubate at 37C incubator for 30 
minutes 
d.Centrifuge for 5 miniutes 
e.Discard supernatent as in step (b) 
f.resuspend in 5ml of fresh fix (3:1 methanol:acetic acid) adding it 
dropwise with good mixing 
g. Store the first fix 12 hours at 4C 
h.Centrifuge & change fix at least twice 
i.Make slides on ethanol washed slides