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Protocols  >  Laboratory protocol  >  Internalization assay

Internalization assay

Laboratory protocol

Phagocytosis assay
Yeasts/spores were biotinylated using 10 mg/ml sulfo-NHS-LC-biotin (Sigma-Aldrich) in 50 mM NaHCO3 pH 8.5 for 2 hours at 4°C. The remaining reactive biotin molecules were inactivated by incubation in 100 mM Tris-HCl pH 8.0 for 40 minutes at 4°C. DCs were then treated with biotinylated spores/yeasts. After 1 hour, cells were permeabilized and labeled with aHLA-DR-FITC. After zymolyase treatment, intracellular yeasts/spores were detected using APC-labeled streptavidin and analyzed by flow cytometry.

Receptor mediated-internalization assay
Immature DCs were allowed to adhere onto fibronectin and subsequently incubated with FITC-labeled yeast or spores for 5, 15, 30 minutes at 37°C. At the end of the incubation period, the samples were fixed in 4% PFA, permeabilized in Methanol, and labeled for DC-SIGN and/or MR using specific mAb and isotype-specific fluorescent secondary Abs. Samples were analyzed using a Zeiss LSM 510 confocal microscope. Alexa647-conjugated goat-anti-mouse IgG, and Alexa568 goat-anti-mouse IgG2b were from Molecular Probes.

Protocol Steps:
  • Phagocytosis assay Yeasts/spores are biotinylated using 10 mg/ml sulfo-NHS-LC-biotin (Sigma-Aldrich) in 50 mM NaHCO3 pH 8.5 for 2 hours at 4°C. The remaining reactive biotin molecules are inactivated by incubation in 100 mM Tris-HCl pH 8.0 for 40 minutes at 4°C. DCs are then treated with biotinylated spores/yeasts. After 1 hour, cells are permeabilized and labeled with aHLA-DR-FITC. After zymolyase treatment, intracellular yeasts/spores are detected using APC-labeled streptavidin and analyzed by flow cytometry.
  • Receptor mediated-internalization assay Immature DCs are allowed to adhere onto fibronectin and subsequently incubated with FITC-labeled yeast or spores for 5, 15, 30 minutes at 37°C. At the end of the incubation period, the samples are fixed in 4% PFA, permeabilized in Methanol, and labeled for DC-SIGN and/or MR using specific mAb and isotype-specific fluorescent secondary Abs. Samples are analyzed using a Zeiss LSM 510 confocal microscope. Alexa647-conjugated goat-anti-mouse IgG, and Alexa568 goat-anti-mouse IgG2b from Molecular Probes.
Internalization assay Graph

created over 8 years ago (21 January 2010)    last modified over 6 years ago (28 September 2011)   [ RDF Rdf ]   [ RelFinder Relfinder ]