Kudriavtsev B N, Kudriavtseva M V, Zavadskaia E E, Shalakhmetova T M, Komarov S A
Tsitologiia. 1979 Jan;21(1):74-83.
A method is proposed for determination of glycogen, DNA, 3H-thymidine incorporation and dry weight in the same cell, the technique being based on successive discovery and measuring of each of these indices. Cells are obtained from animals, previously injected with 3H-thymidine, to be charted on preparation, made pictures and measured in square units. Then on preparations embedded into glycerine or vaseline oil, the optical path difference of rays for the nucleus and cytoplasm of selected cells is measured with the interferencial microscope. This is followed by the fluorescent PAS reaction and the content of glycogen is registered microfluorimetrically in the same cells. Preparations after that are treated with a freshly prepared water solution of 0.025% borohydride sodium, stained with the routine or fluorescent Feulgen reaction, and DNA content is determined in the same cells in which glycogen and delta delta were previously measured. The stained nuclei are photographed, their areas are measured and the dry weight of the nucleus and cytoplasm of marked cells is calculated from the values of the nuclear areas and of delta delta. Eventually the preparations are covered by emulsion and exposed, and 3H-thymidine-containing nuclei are determined, the index of marked nuclei and the marking intensity over the nucleus are calculated. As a result, a precise and reliable determination of glycogen, DNA, dry weight and 3H- or 14C-thymidine incorporation is made in either of the marked cell.
提出了一种在同一细胞中测定糖原、DNA、3H-胸腺嘧啶核苷掺入量和干重的方法,该技术基于对这些指标逐一进行检测和测量。细胞取自预先注射了3H-胸腺嘧啶核苷的动物,制成标本,拍照并以平方单位进行测量。然后,在嵌入甘油或凡士林油的标本上,用干涉显微镜测量所选细胞的细胞核和细胞质的光线光程差。接着进行荧光PAS反应,并用显微荧光法记录同一细胞中的糖原含量。之后,将标本用新配制的0.025%硼氢化钠水溶液处理,用常规或荧光福尔根反应染色,并在先前测量过糖原和光程差的同一细胞中测定DNA含量。对染色的细胞核拍照,测量其面积,并根据细胞核面积和光程差的值计算标记细胞的细胞核和细胞质的干重。最后,将标本覆盖上乳胶并进行曝光,确定含3H-胸腺嘧啶核苷的细胞核,计算标记细胞核的指数和细胞核上的标记强度。结果,在任何一个标记细胞中都能精确可靠地测定糖原、DNA、干重以及3H-或14C-胸腺嘧啶核苷的掺入量。