Direct Fluorescent Labeling of Cells with Fluorescein or Rhodamine Isothiocyanate. I. Technical Aspects

A rapid and simple method of cell labeling by stable conjugation with fluorescein or rhodamine is described. Viable cells are incubated under benign conditions (near physiologic pH in normal media) with free fluorescein or tetramethyl rhodamine isothiocyanate, and are adequately separated from unreacted fluorochrome by washing or centrifugation through fetal calf serum. The effects of the pH, the time and temperature of incubation, and the concentration of cells, fluorochrome, and free protein in the media are described. The method labels all cell types, although to different degrees. Fluorescence microscopy reveals fluorescence throughout the cell, although chromatin appears relatively spared. Cellular fluorescence is fairly stable at 4 and 25 degrees C, decays rapidly at 37 degrees C, but is nonetheless visible for days even at this temperature. In the case of lymphocytes, intense fluorescence is obtained without affecting cell viability, and without alteration of the ability to mount a graft versus host response.