title: Stepwise isolation of human peripheral erythrocytes, T lymphocytes, and monocytes for blood cell proteomics.
abstract: OBJECTIVE
  Density gradient centrifugation and magnetic- or fluorescence-activated cell sorting are common and robust techniques for the isolation of different types of blood cells. In this article, we give detailed description of a stepwise application of these methods as one isolation strategy for enrichment of different cell types from one blood sample.
  
  
  METHODS
  The workflow targeted erythrocytes, monocytes, and T lymphocytes. Pancoll® density gradient centrifugation was used together with subsequent MACS™ isolation. Purity of monocytes and T lymphocytes was controlled by fluorescence-activated cell sorting analysis, and cells were used for carrier-ampholine-based 2D-PAGE to confirm compatibility of the procedure to standard proteomic applications.
  
  
  RESULTS
  Gradient centrifugation resulted in an average of 125 μL of packed erythrocytes per milliliter blood. MACS™ sorting reached purities of 90 ± 2% (monocytes) and 93 ± 2% (T lymphocytes), with an average yield of 12 × 10(4)  monocytes or T lymphocytes. 2D-PAGE of isolated cells showed well-separated spot patterns.
  
  
  CONCLUSIONS
  A combined isolation holds substantial advantages especially in clinical studies, as it allows for the comparison of findings not only between individuals, but also between different cell types derived from one donor. Our approach ensured high reproducibility, yields, and purities of cells as required for reliable proteome analysis.
authors.affiliation: Abteilung für Funktionelle Proteomik, Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Frederic
authors.initials: F
authors.lastname: Brosseron
authors.forename: Caroline
authors.initials: C
authors.lastname: May
authors.forename: Bodo
authors.initials: B
authors.lastname: Schoenebeck
authors.forename: Bettina
authors.initials: B
authors.lastname: Tippler
authors.forename: Dirk
authors.initials: D
authors.lastname: Woitalla
authors.forename: Marion
authors.initials: M
authors.lastname: Kauth
authors.forename: Kathrin
authors.initials: K
authors.lastname: Brockmann
authors.forename: Helmut E
authors.initials: HE
authors.lastname: Meyer
authors.forename: Daniela
authors.initials: D
authors.lastname: Berg
authors.forename: Albrecht
authors.initials: A
authors.lastname: Bufe
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
mesh_terms: D002469:Cell Separation; D002498:Centrifugation; D015180:Electrophoresis, Gel, Two-Dimensional; D004912:Erythrocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D008280:Magnetics; D009000:Monocytes / Q000166:cytology* / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism
chemical_list: D020543:Proteome