|
|
Blood, Vol. 107, Issue 6, 2461-2469, March 15, 2006
Macrophages from C3-deficient mice have impaired potency to stimulate alloreactive T cells
Blood Zhou et al.
107: 2461
Supplemental materials for: Zhou et al
Supplemental Methods Preparation of macrophages. Peritoneal and bone marrow (BM) macrophages were prepared using previously described methods33. In brief, peritoneal macrophages were prepared from normal mice or mice inoculated intraperitoneally (i.p.) with 1ml of 3% thioglycollate 6 days in advance; the peritoneal cell population were harvested and plated on petri dishes to allow macrophages to stick on plastic, then the adherent cells on plastic were collected. Macrophages were further purified with CD11b MicroBeads (Miltenyi Biotec, UK) according to manufacturer’s instruction, unless otherwise specified. BM derived macrophages were cultured from bone marrow progenitor cells. These were harvested from mouse femurs and tibias. In the first 24 hours of culture, BM stromal cells and resident BM macrophages were removed by allowing them to adhere on plastic. The non-adherent cells were continually cultured in macrophage culture medium [EMEM medium, 10% FCS, 100U/ml of Penicillin, 100µg/ml of Streptomycin, 10 µg/ml of macrophage colony-stimulating factor (M-CSF)]. After 7 days, only adherent cells were harvested for the assays. In some experiments, cells were cultured for a further day in the presence of LPS (100µg/ml) to allow the cells to activate. Following the purification, the majority of cells were found to stain positive with anti-mouse F4/80, as determined by flow cytometry. Usually, macrophages were prepared from 3-5 mice and pooled together for the experiments. Thioglycollate-elicited macrophages were used in the majority of our experiments, unless otherwise specified. 3H-thymidine incorporation assay. 2 × 105 irradiated macrophages (H-2b) were added into cultures of 2 × 105 purified primed CD4+ T cells (H-2d) in 96-well plates and cultured for 96 h. 3H-thymidine (1 µCi/well) was added during the last 24 h. The amount of 3H -thymidine incorporation was counted. Control cultures included wells of irradiated macrophages with irradiated T cells. Data were expressed as the difference in cpm of experimental and control cultures. T cells alone and macrophages alone controls were also included in each experiment and gave consistently low backgrounds. Antibody reagents used in Flow cytometry. FITC-conjugated goat anti-mouse C3 IgG F(ab´)2 fragment (ICN Biomedicals, Hampshire, UK), PE-conjugated rat anti-mouse F4/80 (eBioscience, San Diego, USA), PE or FITC-conjugated rat anti-mouse MHC class II (I-A/I-E, M5/114.15.2), PE-conjugated rat anti-mouse CD40 (3/23), PE-conjugated armenian hamster anti-mouse ICAM-1 (CD54, 3E2) and PE-conjugated rat anti-mouse B7.2 (CD86, GL1) (BD Biosciences, Cowley, U.K.). Variance component regression analysis. As the data were repeatedly measured with a nesting structure of repetitions within days within sets, we used variance component regression analysis to test the difference between the two trial groups for each outcome. This model allows us effectively to use all data points, assuming random effects across days and among sets, adjusting for the differences between days and sets. The specific software MLwiN designed for multilevel models was used to conduct this analysis (Rasbash, J., Browne, W., Goldstein, H., et al., 2000. A user’s guide to MlwiN. Institute of Education, University of London).
Files in this Data Supplement:
- Table S1. PCR primer sequences and product sizes (PDF, 17.9 KB)
- Figure S1. Thioglycollate induced macrophages stained with F4/80 (PDF, 10.8 KB)
- Figure S2 (PDF, 23.9 KB) -
Primed alloreactive CD3+ and CD4+ T cells were prepared from BALB/c mice. Thioglycollate-elicited peritoneal macrophages were prepared from WT (C57BL/6), and C3—/— mice. 2 × 105 T cells and 2 × 105 macrophages were co-cultured for 3 days. Supernatants were collected for measuring the production of IL-2 by ELISA. Data depicted were generated from 6 independent experiments and analyzed by Wilcoxon matched pairs test.
- Figure S3 (PDF, 20.8 KB) -
(A) BALB/c mice (n=4) were given either C3+/+ or C3—/— C57BL/6 peritoneal macrophages by i.p. injection. The primed mice then received a C57BL/6 donor skin graft. (B) BALB/c mice (n=4) were primed by skin grafting from C57BL/6 donor and re-challenged with C3+/+ or C3—/— C57BL/6 macrophages. T cell response in those BALB/c mice was then measured by MLR in vitro. The production of IL-2 or/and IFN-
 were measured by ELISA ay day 1-5 after co-culture. Data were analyzed by Two-way ANOVA. P values are for comparisons between two groups.
- Figure S4. MHC staining on BM macrophages (PDF, 20 KB)
- Figure S5 (PDF, 17.9 KB) -
Primed alloreactive CD4+ T cells were prepared from four BALB/c mice. Thioglycollate-elicited peritoneal macrophages were prepared from WT (C57BL/6), C3—/—, C4—/— and FB—/— mice (4 mice in each group). Macrophages generated from each strain of mice were pooled together. 2 × 105 T cells and 2 × 105 macrophages were co-cultured for 3 days. Supernatants were collected for measuring the production of IL-2 and IFN- by ELISA. Data were generated from two independent experiments and analyzed by t test. P values are for comparisons between complement deficient and complement sufficient macrophages. **, P <0.005; ***, P <0.0001; ns, no significant difference.
|
|
