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Blood, Vol. 112, Issue 13, 5084-5094, December 15, 2008
Cyclic AMP plays a critical role in C3a-receptor–mediated regulation of dendritic cells in antigen uptake and T-cell stimulation
Blood Li et al.
112: 5084
Supplemental materials for: Li et al
Files in this Data Supplement:
- Figure S1. C3−/− mouse lymphocytes have impaired responses to Ag re-stimulation measured by 3H-thymidine uptake (JPG, 50 KB)
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WT and C3−/− mice were immunized by i.p. injection with 100 mg of OVA protein in incomplete Freund’s adjuvant. After 10 days (A) and 60 days (B) of immunization, mice were killed. Lymphocytes from lymph nodes and spleen were stimulated with OVA in vitro. Cell proliferation was determined by 3H-thymidine uptake. Each bar represents a single animal and is shown as mean of 6 replicate wells of the ex vivo culture. A representative of two independent experiments is shown.
- Figure S2. The rate of OVA degradation in C3−/− DCs has no apparent defect (JPG, 64.5 KB)
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(A) C3−/− or WT BM DCs were incubated with BODIPYFL dye conjugated OVA (DQ™OVA, a degradation probe) (10 and 100 µg/ml) for 15 minutes and washed with cold PBS for 3 times. The cells were then further cultured in plain medium for up to 30 minutes at 37°C, and OVA degradation was quantified by flow cytometry. The results were generated from duplicate samples and expressed as mean of fluorescence intensity (MFI). Data are shown as mean of duplicate samples. A representative of two independent experiments is shown. (B) Day 7 WT DCs were fixed with formalin/gluteraldehyde and processed as sucrose-infused cell sections. The cell sections were incubated with goat anti-mouse IgG C3 followed by 10nm gold-conjugated rabbit anti-goat IgG, prior to imaging by electron microscopy. Small black dots represent C3. Images are views of parts of two different cells. The darker areas represent electron-dense cell cytoplasm, while the lighter areas constitute the extracellular space (ECS) or endocytic vesicles (EV) within the cells.
- Figure S3. The C3a treatment has no apparent effect on Ag uptake and surface molecule expression in C3aR−/− DCs (JPG, 40.4 KB)
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C3a (20 nM) was added into DC culture medium from the beginning of BM cell culture up to 7 days. (A) Ag uptake performed in 6 day DCs. Data are shown as mean ± SEM (n=4). (B) Surface expression of MHC class II (MHC-II), CD40 and B7.2 in 7 day DCs by flow cytometry. Data are shown as mean ± SEM (n=3). Data were analysed by Student’s t test. ns. no significant difference. A representative of two independent experiments is shown.
- Figure S4. Effect of inhibition of MAPK and PI3K pathways on DC cytokine profile (JPG, 62.2 KB)
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Effect of inhibition of AKT, ERK and P38 pathways on cytokine secretion. 6 day DCs were further cultured for 24 h in the presence of absence of the appropriate inhibitor i.e. wortmannin (for AKT), U0126 (for ERK), SB202190 (for P38). The supernatants were used for cytokine measuring by ELISA. Data are shown as mean ± SEM (n=4). A representative of two independent experiments is shown.
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