[Cytophotometric determination of non-heme iron content in hepatocytes. I. Effect of cell separation techniques on...

To study conditions of the preservation of non-heme iron (3+) in hepatocytes, experiments were performed on rats fed with the diet supplemented with 2% carbonyl iron. The cells were isolated by a collagenase method (an enzymatic method) or by a treatment of the tissue with the phosphate buffer (a nonenzymatic method). When using the enzymatic method, the main steps of obtaining preparations-smears were analysed: perfusion of the organ, subsequent washing out of the cells from collagenase, mounting of the smear on the object glass. When using the non-enzymatic method, such steps were an incubation of the tissue pieces in the isolation solution and mounting of the smears. It has been found that the enzymatic isolation method results in practically no losses of iron from the cells if the perfusion lasts for 20 min. A slight loss (10-12%) of the Perls'-stained iron can occur during the initial 30 min of the washing out of the cells from collagenase. This step is not accompanied by any morphological changes of the cells; their viability, according to the trypan test, is 70-87%. The iron release from the cells rises with decrease in the viability of hepatocytes. It has been shown that the greatest losses of iron can occur at the mounting step when the cells are submitted to a substantial mechanical effect. When the nonenzymatic method is applied, the incubation of the cells in the phosphate buffer for up to 2 hr causes no marked morphological changes revealed in the light microscope; however, the cell viability is very low (about 1%). The preservation of iron in the cells is lower when using the nonenzymatic than the enzymatic method. Thus, for cytophotometric determinations of the iron content in hepatocytes, the collagenase method of isolation of cells is recommended.