Ova alexa647

To measure antigen (Ag) uptake, DCs (3×10⁵/sample) were left untreated, or pretreated with LPS (100 ng/ml) and 10 ng/ml of CyaA or CyaA-AC⁻ for 30 min. Subsequently, OVA-Alexa647 or transferrin-Alexa647 (both 5 µg/ml, Invitrogen) or Lucifer yellow (500 µg/ml, Invitrogen) were added for 30 min at 37°C. The Ag uptake was assessed by flow cytometry.
For MHC class I-restricted processing DCs (2×10⁶/sample) were left untreated, or incubated with 10 ng/ml of CyaA or CyaA-AC⁻ ng/ml or lactacystin (10 µM, Sigma-Aldrich) and LPS (100 ng/ml) for 30 min. Protein concentration in lysate was determined by MicroBCA™ Protein Assay kit (Pierce). 50 µg of proteins in 20 mM Tris-HCl, pH 7.4 was mixed with 100 µM Z-Leu-Leu-Glu-AMC, Suc-Leu-Leu-Val-Tyr-AMC or Boc-Leu-Arg-Arg-AMC fluorogenic substrates (BIOMOL) and incubated for 90 min at 37°C. The fluorescence of liberated 7-amino-4-methylcoumarin was measured using a microplate reader (380_ex/460_em, Safire², Schoeller Instruments).
For MHC class II-restricted Ag processing DCs (1×10⁶/sample) were left untreated, or incubated with 10 ng/ml of CyaA or CyaA-AC⁻ or chloroquine (100 µM, Sigma-Aldrich) and LPS (100 ng/ml) for 30 min. DCs were subsequently loaded with a mixture of OVA-Alexa647 and OVA labeled with BODIPY FL dye (OVA-DQ, both 5 µg/ml, Invitrogen) for 30 min at 37°C and analyzed by flow cytometry [32] (link).

Mice were immunized i.v. with OVA (Grade V; Sigma-Aldrich) or OVA-Alexa 647 (Life technologies, Carlsbad, CA) alone, or with IgE anti-OVA pre-mixed with OVA or OVA-Alexa 647 in 200 μl PBS. The doses used are indicated in each figure.