title: Pitfalls in flow cytometric analyses of surfactant-exposed human leukocytes.
abstract: BACKGROUND
  Surfactant replacement treatment is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome in preterm infants and may also improve oxygenation in acute respiratory distress syndrome in children, adolescents and adults. Beside surface tension- and mechanical shear-reducing functions, natural surfactants have been ascribed immunomodulatory capacities. Current in vitro studies on immunomodulatory effects of pulmonary surfactant preparations on human leukocytes rely on ELISA, Western blot and polymerase chain reaction. Data obtained by flow cytometry are missing, so far, most likely due to confounding phospholipid residues. Intracellular cytokine flow cytometry in surfactant-exposed immune cells would provide information on pro- and anti-inflammatory immunomodulation at the single-cell level and would allow for integrating detailed immunophenotyping, functional assays and assessment of viability.
  
  
  OBJECTIVE
  We implemented a flow cytometry protocol for reliable quantitative assessment of in vitro intracellular cytokine production in surfactant-exposed human lymphocytes (CD4(+)) and monocytes (CD14(+)).
  
  
  METHODS
  Two different permeabilization techniques were tested for their ability to provide intracellular cytokine staining in surfactant-exposed CD14(+) monocytes and CD4(+) lymphocytes. Both a commercially available solution containing saponin and ice-cold methanol were used as permeabilization reagents.
  
  
  RESULTS
  For both cell types, flow cytometry following saponin-based permeabilization revealed pronounced unspecific fluorescence signals in surfactant-exposed samples overlapping with the fluorescence spectra of the majority of conjugates. Autofluorescence of surfactant phospholipid particles interfered significantly with reliable quantification of fluorochrome-specific signals and conclusive analysis. Implementation of a methanol-based permeabilization protocol resulted in the elimination of confounding non-cell particle signals allowing for an accurate quantification of intracellular cytokine production.
  
  
  CONCLUSIONS
  Reliable detection of intracellular cytokines by flow cytometry may be challenging in surfactant-exposed cell samples due to significant autofluorescence of aggregated phospholipid particles. This issue has been addressed for the first time and may be of high relevance for all types of surfactant research. We demonstrate that a methanol-based permeabilization approach completely removes interfering fluorescent surfactant micelles and allows for correct evaluation of data. The successful removal of confounding surfactant phospholipids opens up a wide variety of multiparameter flow cytometry; a method that has not been applied in the field of surfactant research, yet.
authors.affiliation: University Children's Hospital, University of Wuerzburg, Wuerzburg, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Glaser
authors.affiliation: University Children's Hospital, University of Wuerzburg, Wuerzburg, Germany. Electronic address: markus.fehrholz@uni-wuerzburg.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Fehrholz
authors.affiliation: University Children's Hospital, University of Wuerzburg, Wuerzburg, Germany.
authors.forename: Silvia
authors.initials: S
authors.lastname: Seidenspinner
authors.affiliation: University Children's Hospital, University of Wuerzburg, Wuerzburg, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Ottensmeier
authors.affiliation: University Children's Hospital, University of Wuerzburg, Wuerzburg, Germany.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Wollny
authors.affiliation: University Children's Hospital, University of Wuerzburg, Wuerzburg, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Kunzmann
mesh_terms: D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D056747:Immunomodulation / Q000187:drug effects; D016130:Immunophenotyping; D007231:Infant, Newborn; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D011663:Pulmonary Surfactants / Q000494:pharmacology*; D012128:Respiratory Distress Syndrome / Q000628:therapy; D012127:Respiratory Distress Syndrome, Newborn / Q000628:therapy; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D011663:Pulmonary Surfactants
keywords: Autofluorescence; CD14(+) monocytes; CD4(+) T cells; Curosurf(®); Flow cytometry; Phospholipids; Surfactant