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Figure S1. Control experiment to check the efficacy of MG132 in blocking direct presentation of cytosolic OVA. Both live and fixed cells were incubated with a hypertonic solution of OVA and subjected to a hypotonic shock.

Figure S2. Estimation of cross-presentation yield. (A) Calibration of the fluorescence intensity of an Alexa Fluor 647 F(ab′)2 fragment using QIFIKIT beads loaded with known numbers of mouse epitopes. Fluorescence was determined by flow cytometry. (B) Standard curve derived by plotting mean fluorescence intensity of the stained beads against the number of epitopes per bead. (C) Staining of SIINFEKL-MHC class I complexes on the surface of DC2.4 cells loaded with varying concentrations of exogenous SIINFEKL using the 25-D1.16 antibody. The number of complexes was calculated using the standard curve in (B). (D) Response of B3Z cells to DC2.4 cells loaded with varying concentrations of exogenous SIINFEKL. There is a degree of overlap between the upper range of the B3Z response curve and the lower range of sensitivity of the Kb-SIINFEKL-specific antibody. This overlap was used to translate B3Z response into Alexa Fluor 647 fluorescence intensity.

Figure S3. Distribution of actin around phagocytosed MS. DC2.4 cells were fed yellow-green fluorescent MS shown in green for 30 min, before fixing and staining cellular actin with a phalloidin-Alexa Fluor 647 conjugate (shown in red). Upper panels: cells were fed sonicated 3.0 μm MS. Lower panels: cells were fed aggregated 0.8 μm MS. Actin: single optical sections showing actin alone. Actin, MS: as before, but with the fluorescent MS overlaid. Actin z-stack: actin maximum projection of entire cells. Actin, MS z-stack: as before, but with the fluorescent MS overlaid. DIC: differential interference contrast image. Merge: Superimposition of fluorescence and DIC images. Arrows point to examples of phagosomes surrounded by actin. White bars represent 10 μm.

Figure S4. Translocation of c-Fos from cytoplasm to nucleus after phagocytosis of microspheres. 0.8 μm: DC2.4 cells were fed sonicated 0.8 μm MS according to Experimental Procedures. Cells were fixed and stained 1 hr (upper panels) or 4 hr (lower panels) after the start of the 30 min antigen pulse. C-Fos is shown in green and the nucleus in red. Areas of colocalisation appear yellow. DIC: differential interference contrast image. Merge: overlay of fluorescent and DIC images. 3.0 μm: cells were fed sonicated 3.0 μm MS. 0.8 μm Agg: cells were fed aggregated 0.8 μm MS. White bar represents 10 μm.

Video 1. Time-lapse animation of DC2.4 cells incubated for 24 hr in the absence of microspheres.

Videos 2–4. Time-lapse animations of DC2.4 cells incubated for 24 hr in the presence of OVA-coated 3.0 μm MS.

Data S1. Detailed Materials and methods.

FilenameFormatSizeDescription
IMM_3558_sm_FigS1.PDF21KSupporting info item
IMM_3558_sm_FigS2.PDF139KSupporting info item
IMM_3558_sm_FigS3.PDF1614KSupporting info item
IMM_3558_sm_FigS4.PDF2118KSupporting info item
IMM_3558_sm_Supplemental-Methods.doc34KSupporting info item
IMM_3558_sm_Video-3.avi5041KSupporting info item
IMM_3558_sm_Video-4.avi3106KSupporting info item
IMM_3558_sm_Video-1.avi3863KSupporting info item
IMM_3558_sm_Video-2.avi5037KSupporting info item

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