David LePage's Gene Targeting Protocols

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Counting ES cell Chromosomes

(original reference: "tissue culture made easy" by Christian LaMantia from the Magnuson lab)

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). 2ml per 35 mm and 4ml per 60 mm.

3) Remove media and place it in a 15 ml Falcon tube. Wash with trypsin, and trypsinize with 1.5 ml trypsin.

4) Centrifuge on 4 for 5 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 incubate in 37°C water bath for 10 minutes.

6) Centrifuge on setting 4 for 5 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. Can be kept in the fridge at this point forever, then processed for spreads when convenient.

8) Spin down and aspirate old fix with pasteur pipet. Resuspend cells in 0.5 to 0.75 ml of fresh fix and gently draw suspension up in a pasteur pipet(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. Drop 3 drops of cell suspension per slide and air dry. Drops should make a gentle splat from a height of approximately one foot above the slide.

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 3-4 times until the excess stain is removed. Air dry.

Five spreads on 4 slides give a reasonable sampling. Scan slides with a 40 x objective and count with 100 x objective. Look at spreads in widely separated fields. Spreads with fewer than 40 chromosomes are ignored as probable splattered cells. Lines with 41 or 42 chromosomes are the usual problem.


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