Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI

Background: This study investigated the relationship between chromatin comp actness, which is directly related to chromatin condensation, and DAPI upta ke. Materials and Methods: For the structural characterization of in situ chrom atin, we used fluorescence microscopy and differential scanning calorimetry on calf thymocytes. The compactness of nuclear chromatin was altered by pe rmeabilizing native cells with NP40 detergent. A time-dependent analysis of detergent effects was performed by acquiring nuclear images at different t ime intervals after permeabilization. In order to compare nuclei of differe nt sizes, we implemented a geometrical correction in the calculation of the integrated fluorescence intensity. For a quantitative evaluation of chroma tin condensation we introduced two new parameters, "average chromatin packi ng ratio" and "average: dye spatial density.'' Results: This approach allowed us to estimate the effects of NP40 detergent at the level of in situ chromatin. Detergent effects could be modulated by changing the ionic composition of buffer. Moreover, changes of chromatin c ondensation induced by detergent were inversely related to modifications of nuclear volume. Conclusions: The combination of complementary information obtained by fluor escence microscopy, supported by a proper geometrical correction, and diffe rential calorimetry allowed us to interpret the patterns of fluorescence in tensities inside the nucleus in terms of chromatin structure. Cytometry 44: 113-119, 2001. (C) 2001 Wiley-Liss, Inc.