[DNA flow cytometric evaluation of spermatogenesis. Part 1: Analysis of nuclear DNA in cells from the human testicular tissue].
The present study was carried out to establish the best method of preparing human testicular tissue for flow cytometric DNA analysis including dispersal, fixation and staining. Human testicular tissue could be dispersed to single cells by incubating in 0.05% collagenase solution at 37 degrees C for 60 minutes. Krishan's method which stains nuclear DNA directly without ethanol fixation and digestion in ribonuclease was not suitable for testicular cells. After ethanol fixation, testicular cells were treated with ribonuclease and pepsin, then stained with propidium iodide. Nuclear DNA in cells was measured by flow cytometry and a good DNA histogram was obtained. Ribonuclease influenced the DNA histogram little, but pepsin markedly improved it by digesting cell debris and decreasing cell aggregation. Analysis of the DNA histogram revealed the proportion of haploid, diploid and tetraploid cells accurately and quickly. Flow cytometric DNA analysis could be a useful method of evaluating cell kinetics in spermatogenesis.
D002454:Cell Differentiation; D002469:Cell Separation; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008297:Male; D013091:Spermatogenesis; D013737:Testis / Q000032:analysis* / Q000166:cytology
D004247:DNA