title: Rapid isolation of nuclei from living immune cells by a single centrifugation through a multifunctional lysis gradient.
abstract: 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 or contamination 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 from primary 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-containing lysis 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 lysis gradient 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 15 min with the ability to handle as little as one million cells per sample and easy parallel processing of multiple samples.
authors: Department of Internal Medicine III, Division of Nephrology and Dialysis, Medical University Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
authors: Marko
authors: M
authors: Poglitsch
authors: Karl
authors: K
authors: Katholnig
authors: Marcus D
authors: MD
authors: Säemann
authors: Thomas
authors: T
authors: Weichhart
mesh_terms: D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000378:metabolism*; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014283:Triiodobenzoic Acids / Q000737:chemistry
chemical_list: D008070:Lipopolysaccharides; D014157:Transcription Factors; D014283:Triiodobenzoic Acids; C044834:iodixanol