A protocol to quantify chromatin compaction with confocal and super-resolution microscopy in cultured cells

Abstract

Here, we describe three complementary microscopy-based approaches to quantify morphological changes of chromatin organization in cultured adherent cells: the analysis of the coefficient of variation of DNA, the measurement of DNA-free nuclear areas, and the quantification of chromatin-associated proteins at the nuclear edge. These approaches rely on confocal imaging and stochastic optical reconstruction microscopy and allow a fast and robust quantification of chromatin compaction. These approaches circumvent inter-variability between imaging conditions and apply to every type of adherent cells. For complete details on the use and execution of this protocol, please refer to Neguembor et al. (2021). ; The authors acknowledge the support from European Union's Horizon 2020 Research and Innovation Programme (CellViewer no. 686637 to M.P.C. and M.L.); Ministerio de Ciencia e Innovación, grant (BFU2017-86760-P [AEI/FEDER, UE] to M.P.C.), and an AGAUR grant from Secretaria d'Universitats i Recerca del Departament d'Empresa i Coneixement de la Generalitat de Catalunya ([2017 SGR 689] to M.P.C.); Centro de Excelencia Severo Ochoa (2013–2017 to M.P.C.); CERCA Programme/Generalitat de Catalunya (to M.P.C.); the Spanish Ministry of Science and Innovation to the EMBL partnership (to M.P.C.); National Natural Science Foundation of China (31971177 to M.P.C.); Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110103001 to M.P.C.); People Program (Marie Curie Actions) FP7/2007–2013 under REA grant 608959 (to M.V.N.); Juan de la Cierva-Incorporación 2017 (to M.V.N.); grant for the recruitment of early-stage research staff FI-2020 (Operational Program of Catalonia 2014-2020 CCI 2014ES05SFOP007 of the European Social Fund to L.M.); "La Caixa" Foundation Fellowship (ID 100010434, #LCF/BQ/DR20/11790016) (to L.M.); and "La Caixa-Severo Ochoa" pre-doctoral fellowship (to A.C.-G.)