[The variability of the pattern of human G-band chromosomes in the late prophase-early metaphase].
Original modification of the harvest procedure, hypotonic treatment and slide-making techniques were used to obtain prometaphase spreads of good quality fitted to G-banding or to FISH. Human blood cultures were synchronized with a methotrexate block during synthesis, and following this with a thymidine release. Cells were then proceeded into prometaphase and early metaphase and were quickly harvested without exposure to colcemid. Following incubation in a mixture of 0.56% KCl and 1% sodium citrate (1:1) for 20-23 min at room temperature, cells were fixed in 4 changes of fixative made of 3 parts of absolute methanol and 1 part of glacial acetic acid. Three or four 100-105 microliters volume of a suspension of fixed cells were blew with a strength through pasteur pipette tip from a distance 5-6 cm onto chilled superclean slides wetted by water. These slides were then quickly flame dried. Prior to staining, prometaphase bearing spreads were held in thermostat overnight at 37 degrees C. Prometaphases were G-banded with the aid of a modified technique elaborated in the Department of Clinical Genetics, Lund University Hospital (Sweden). The slides were incubated for 1-4 min at room temperature in 0.01 versene solution, containing 0.011% w/v trypsin (Difco), 0.4 mg/ml D-glucose, 0.17 mg/ml KCl, 1.7 mg/ml NaCl. Following a 15 sec incubation in a solution containing 1 mg/ml D-glucose, 0.4 mg/ml KCl, 8 mg/ml NaCl, G-banding was completed by staining the slides with 2% Giemsa (Merck) in pH 6.8 phosphate buffer for 4-5 min.(ABSTRACT TRUNCATED AT 250 WORDS)
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D000328:Adult; D002478:Cells, Cultured / Q000648:ultrastructure; D002871:Chromosome Banding / Q000379:methods*; D014644:Genetic Variation; D006801:Humans; D008214:Lymphocytes / Q000648:ultrastructure; D008677:Metaphase; D008875:Middle Aged; D011418:Prophase; D013997:Time Factors
Izmenchivost' risunka G-okrashennykh khromosom cheloveka v pozdneĭ profaze--ranneĭ metafaze.