Nucleo

Rapid isolation of nuclei from living immune cells by a single centrifugation through a multifunctional lysis gradient.
Due to their low protein content and limited nuclear detergent stability,primary human immune cells such as monocytes or T lymphocytes represent a great challenge for standard nuclear isolation protocols. Nuclei clumping during the multiple centrifugation steps orcontamination of isolated nuclei with cytoplasmic proteins due to membrane lysis is a frequently observed problem. Here we describe a versatile and novel method for the isolation of clean and intact nuclei fromprimary human monocytes, which can be applied for virtually any cell type. Living cells were applied on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containinglysis layer. Mild cell lysis as well as efficient washing of the nuclei was performed during the course of one single low g-force centrifugation step. The isolation procedure, which we call lysisgradient centrifugation (LGC), results in the recovery of 90-95% of highly pure nuclei. This easy and highly reproducible procedure allows an effective preparation of nuclei and the cytoplasm in only 15minwith the ability to handle as little as one million cells per sample and easy parallel processing of multiple samples.
http://www.ncbi.nlm.nih.gov/pubmed/21889513

Debido a su bajo contenido enproteínas y poca estabilidad detergente nuclear, las células humanas primarias inmunológico, como los monocitos o linfocitos T representan un gran desafío para los protocolos estándares de aislamientonuclear. Los núcleos de aglomeración durante los pasos de centrifugación múltiple o la contaminación de los núcleos aislados de proteínas citoplasmáticas debido a la lisis de membrana es un problemafrecuentemente observado. Aquí se describe un método versátil y novedosa para el aislamiento de los núcleos limpios e intactos de primaria monocitos humanos, que se puede aplicar a prácticamente...