1) Plate cells onto gelatinized plates (35 or 60 mm) without feeders and culture 24 hours. A 1:4 split is best for most lines.
2) Culture for four hours in the presence of colcemid (3.125 µl of 10 µg/ml colcemid (Gibco # 15212-012) per ml of medium).
3) Remove media, wash with STV, and trypsinize with 1.5 ml STV.
4) Centrifuge on 4 for 4 minutes. Aspirate supernatant and flick pellet.
5) Add 37°C KCl (0.559 g KCl in 100 ml water) drop by drop, flicking the pellet with each drop for the first 10-15 drops. Bring volume up to 7 ml, invert several times, and incube in 37°C water bath for 10 minutes.
6) Centrifuge at 5 in clinical centrifuge for 4 minutes.
7) Aspirate supernatant and flick pellet. In the next step, be careful to disperse the cells thoroughly in the fix. Add fresh fix (2 ml glacial acetic acid and 5 ml methanol) drop by drop, flicking the pellet with every drop for the first 10 to 15 drops. Cap and incubate at 4°C overnight.
8) Spin down and aspirate old fix with pasteur pipet. Resuspend cells in 0.5 to 0.75 ml of fresh fix (will make 4 to 5 slides).
9) Dip slides (plain, pre-cleaned slides (Fisher #12-549) labelled with etching tool) in water, leaving a puddle of water on the slide. From one foot above the slide, drop 3 drops of cell suspension onto each slide and air dry.
10) Stain for 10 minutes in Coplin jar with Giemsa (2.5 ml of Giemsa (Gibco #10092-013) in 47.5 ml of Gurr’s pH 6.8 buffer (Gibco #10582-013)).
11) Rinse with water in Coplin jar until clear. Air dry.
Five spreads on 4 slides give a reasonable sampling. Scan slides with a 40X objective and count with 100X objective. Look at spreads in widely separated fields. Spreads with fewer than 39 chromosomes are ignored as probable splattered cells. Lines with 41 chromosomes are the usual problem.
A Conlon Lab Protocol
pdf file for this protocol