DEOXYRIBONUCLEIC ACID CYTOPHOTOMETRY OF STAINED HUMAN LEUKOCYTES III. THERMAL DENATURATION OF CHROMATIN

Abstract

Thermal denaturation of chromatin has been monitored in situ in single cells using absorption cytophotometry on gallocyanin-chrome alum (GCA)-stained preparations. Spreads of human lymphocytes were fixed by ethanol freeze-substitution, digested with ribonuclease and treated at different temperatures prior to staining. Treatment in 0.15 M NaCl-0.0l5 M Na citrate (pH 7.0) for 10 min was followed by 0.15 M NaCl-0.0l5 M Na citrate (pH 7.0) with 10% formalin at the same temperature for 20 min. The GCA staining was localized to nuclei and was measured cell by cell with an integrating scanning cytophotometer. Cellular GCA stain content increased progressively with treatment temperatures above 60°C until 90°C, when it was about 1.5 times that of cells treated at 22°C. Formation of single strand deoxyribonucleic acid at different temperatures was monitored by digesting slides with an endonuclease that is specific for single strand nucleic acids and then staining with GCA. Digestion had little effect on slides that had been treated at less than 70°C, but above this temperature it caused a progressive decrease in stain content up to 90°C, when the stain content was reduced to about one-half that of undigested cells. Both with and without enzymatic digestion, these new approaches to monitoring chromatin denaturation yield preparations that are stable and can be measured on any visible light absorption cytophotometer; in addition, the enzymatic approach exemplifies a class of potential methods in which biochemical procedures are adapted to cytophotometry.