Quantitative single cell methods that identify cytokine and chemokine expression in dendritic cells

Karin Loré, Anna Lena Spetz, Thomas E. Fehniger, Anders Sönnerborg, Alan L. Landay, Jan Andersson

Research output: Contribution to journalArticlepeer-review

Abstract

Two techniques based upon flow cytometry (FCM) and in situ image analysis were developed for quantification of intracellular cytokine and chemokine protein expression at the single cell level in dendritic cells (DCs). The qualitative and quantitative differences between the two methods were evaluated. In vitro differentiated DCs were stimulated with lipopolysaccaride (LPS) and thereafter stained for either IL-8, which is secreted through the Golgi-organelle, or IL-1ra, which localises diffusely in the cytoplasm. Microscopic examination, both for fluorophore and enzymatically stained cells, showed that DCs expressed IL-8 and IL-1ra with two different staining patterns. FCM analysis showed high frequencies of IL-1ra producing cells (76±13%), which was similar to the frequency obtained by in situ imaging. However, in contrast to IL-1ra, the incidence of IL-8 expressing DCs showed high variability between the donors. The numbers of positive cells were 19±19% as measured by FCM. The detection of IL-8 analysed by in situ imaging revealed higher frequencies (26±14%). The addition of brefeldin-A, leading to cytoplasmic accumulation of proteins secreted through the Golgi endoplasmatic route, generated a significantly increased signal intensity and incidence of producer cells, resulting in similar frequencies for both methods. FCM has the advantage of being less time consuming than image analysis and is also able to facilitate multiple colour analysis. However, FCM is less accurate in detecting and quantifying cytokines and chemokines with a preserved juxtanuclear staining pattern. The correct choice of detection technique therefore depends on the study question.

Original languageEnglish (US)
Pages (from-to)207-222
Number of pages16
JournalJournal of Immunological Methods
Volume249
Issue number1-2
DOIs
StatePublished - Mar 1 2001
Externally publishedYes

Keywords

  • Chemokines
  • Cytokines
  • Dendritic cells
  • Flow cytometry
  • In situ imaging analysis

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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