Methods for improving the yield and quality of metaphase preparations for FISH probing of human lymphocyte chromosomes.
Procedures are described for the in vitro culture of human lymphocytes, which have been concentrated by density gradient centrifugation, and for a modified slide-making technique for the fixed cells. The method yields improved percentages of mitotic cells which are largely synchronized at harvest. Controlled placement of fixed cells on slides produces well-spread metaphase preparations with little background material to interfere with fluorescence in situ hybridization (FISH) probe procedures. The FISH reagents and microscope scanning time required are minimized by concentrating cells in a defined area of the slide.
McFee
A F
AF
Environmental and Health Sciences Division, Oak Ridge Institute for Science and Education, Tennessee 37831-0117, USA.
Sayer
A M
AM
Salomaa
S I
SI
Lindholm
C
C
Littlefield
L G
LG
D018929:Cell Culture Techniques / Q000379:methods*; D015925:Cryopreservation; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008214:Lymphocytes / Q000166:cytology*; D008677:Metaphase; D008940:Mitotic Index; D015203:Reproducibility of Results; D013048:Specimen Handling; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors