Cytochemical fractionation of the Lettre-Ehrlich ascites tumor.

SUMMARY A method for cytochemical fractionation of the Lettré-Ehrlich ascites tumor has been presented. The cells were exposed to dilute Ringer solution which increased the cell size and rigidity and allowed them to be homogenized in 0.44 M sucrose with the use of the Dounce all-glass "ball and cylinder" homogenizer. Nuclear, mitochondrial, and microsome + "soluble" fractions were obtained and assayed for cytochrome oxidase and peroxidase activities. A good nuclear fraction was obtained from this tumor as shown by microscopic examination and as indicated by the low cytochrome oxidase content. The mitochondrial fraction was evaluated only by enzymatic meth ods. As would be expected, both cytochrome oxidase and peroxidase occurred pre dominantly in the mitochondrial fraction. Spectral data indicated that the predominant pigment of the mitochondria was cvtochrome c. The technic of tissue homogenization and dif ferential centrifugation is now widely used. Most animal tissues yield to this technic with ease, and large amounts of cellular particulates can be quickly obtained. Ascites tumor cells, however, have tended to resist successful homogenization by the methods in common use, since the membrane and matrix of these cells are especially strong and cannot be ruptured by procedures designed for liver cells (9). This has resulted in methods with media of high viscosity (1, 15), cellular disruption by shaking with quartz sand (9), lysis with digi-tonin (8), and a mechanical device (5). The me chanical methods require equipment that is elabo rate and costly for small preparations. The technic of shaking with quartz sand has, in this laboratory, given low yields of homogenate and requires subse quent separation of the sand. The method with high viscosity media yields only a well defined nu clear fraction. The studies to be presented in this paper de scribe a method for homogenization of the Lettré-Ehrlich ascites tumor cells that yields from the same homogenate nuclear and mitochondrial frac tions as well as a supernatant fraction designated as the microsomal + "soluble" fraction. The asci-tes cells were suspended in quarter strength ascites Ringer solution in preparation for homogenization. The homogenizer used was the all-glass "ball and cylinder" type described by Dounce (4), which can be operated manually. The distribution of cyto chrome oxidase and peroxidase in the cell particu lates is also presented. Tumor transplantation and harvest.†" Male Swiss Ha/ICR strain mice, between the ages of 8 weeks and 6 months, were given implants intraperi-toneally, by syringe, …