|
|
Blood, Vol. 111, Issue 4, 2452-2461, February 15, 2008
Local production and activation of complement up-regulates the allostimulatory function of dendritic cells through C3a–C3aR interaction
Blood Peng et al.
111: 2452
Supplemental materials for: Peng et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 66.7 KB)
- Table S1. PCR primer sequences and product sizes (PDF, 59.3 KB)
- Figure S1. Conventional RT-PCR was performed with cDNA prepared from 6 day-BM DCs and spleen or liver tissue (JPG, 36.6 KB)
-
The typical agarose gels show the PCR products for DAF, Cr2, fH, C2, C4, C5, C6, C8, C9 and GAPDH (internal control). The 100 bp DNA markers are shown along side the gels. All results are representative of at least three independent experiments.
- Figure S2 (JPG, 37.9 KB)
-
(A) Detection of fB, Crry and C1q by western blot. The supernatants were collected at d2, d4 and d6 of BM DC culture, resolved by SDS-PAGE on a 4-20% Tris glycine gradient (fB and Crry) or 12% gels (Invitroge) and transferred to nitrocellulose membrane. Blots were blocked for 1 h with 5% skim milk in TBST and then incubated with primary antibodies goat anti-human fB (Quidel, 1:1000) and anti-mouse Crry (Gift from Dr M Holers, 1:2000) for 1 h, or rabbit anti-mouse C1q (Hbt, 1:200) at 4°C overnight. The blots were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulin (Dako, 1:1000) for 1 h. Immunoreactive proteins (92 kDa for fB, 65 kDa for Crry and ∼25 kDa for C1q) were detected with enhanced chemiluminescence (Amersham Biosciences) and exposed to Kodak X-Omat Film. Human (for fB) and mouse (for C1q) serum (1: 100 dilution) were used for positive controls. (B) Detection of CR3, CR4 and C5aR on BM DCs by flow cytometry. The cultured 6-day BM DCs were pre-incubated with Fc-blocking antibody. 2 × 105 DCs were stained with PE or APC-conjugated rat anti-mouse antibodies (CD11c or CD11b, respectively) or with biotinylated rat anti-mouse antibody (C5aR) and FITC-conjugated goat anti-rat Ig or the appropriate isotype control antibodies, at 4°C for 30 min followed by washing 3 times in 2 ml of PBS containing 1% BSA. The cells were then fixed in 400 µl of 1% paraformaldehyde in PBS. The stained cells were analysed by flow cytometry and represented by the open peaks. Isotype controls are represented by the solid peaks. A representative of three independent experiments is shown.
| |
