Leukaemia cytogenetics / H. Jackson Woodliff.

  • Woodliff, H. Jackson.
Date:
1971
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Leukaemia cytogenetics / H. Jackson Woodliff. Source: Wellcome Collection.

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    METHODS 11 tation rate (common in leukaemia) is collected into a sterile syringe from a cubital vein and 10 ml placed in a sterile universal bottle containing 1 mg of heparin dissolved in 0-1 ml of distilled water. Blood from patients without a high ESR is collected similarly and added to a bottle containing 0-5 ml of sterile dextran solution (Dextraven—6 per cent dextran in saline) in addition to the heparin. After mixing, the specimens are allowed to stand at room temperature for half an hour to two hours, the leucocyte rich super¬ natant plasma is removed and a cell count performed; the remain¬ der of the specimen is centrifuged to produce cell free plasma. Where necessary, the cell count is adjusted to 5-10,000 leuco- cytes//xl by the addition of patients' cell free plasma or, if unavail¬ able, with sterile AB plasma or serum. The cell suspension is then diluted with four volumes of tissue culture fluid 199 (Difco M) or Eagle's basal medium, and 0-1 ml of a PHA M (Difco) solution, prepared according to the direction of the manufacturers, is added to each 5 ml of culture fluid if mitotic stimulation is required. Depending upon the material available and the purpose of the culture, a number of aliquots are put up in 2 to 10 ml amounts in 15 ml screw-capped bottles and when necessary the pH is adjusted to between 7-0 and 7-2 by the addition of 5 per cent carbon dioxide in air. Cultures are incubated at 37°C and harvested at approxi¬ mately 24, 48 and 72 hours after prior treatment of two to four hours with demecolcine to a ñnal concentration of 2 j^tg/ml. On harvesting, cell clumps are broken up by pipetting and the culture transferred to a suitable tube forcentrifuging for five to ten minutes; the supernatant is then discarded and the cells washed with Hank's solution. After again centrifuging, the button of cells is suspended in 2 ml of Hank's solution which is then diluted with 6 ml of pre-warmed 37°C distilled water. After thoroughly mixing, the cells are incubated for a further five minutes after which they are centrifuged at low speed for 10 minutes and all but 2 ml of the supernatant removed. 3 ml of freshly made fixative (3 volumes methanol, 1 volume glacial acetic acid) are layered on top of the aqueous phase and this is gently removed from beneath the fixative with a Pasteur pipette so that the methanol and glacial acetic acid bathe the button of cells at the bottom of the tube. The cell button is then broken up to make a uniform suspension by gently pump¬ ing with a pipette, and the cells are again centrifuged, washed with fresh fixative twice and finally suspended in about 0-5 ml of fresh fixative. Three drops of the suspension are dropped onto a water