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Blood, Vol. 113, Issue 8, 1759-1767, February 19, 2009
B7-H4–deficient mice display augmented neutrophil-mediated innate immunity
Blood Zhu et al.
113: 1759
Supplemental materials for: Zhu et al
Files in this Data Supplement:
- Table S1. Absolute number of neutrophils in spleens prior and post Listeria Monocytogene infection# (PDF, 15 KB)
- Figure S1. Decoy B7-H4 enhances neutrophil response to LPS in B6 mice (JPG, 19.5 KB)
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To generate decoy B7-H4V and B7-H4VC plasmids, 2 primers flanking 5 and 3′ of IgV fragment of murine B7-H4 were designed with XhoI and EcoRI restriction sites, respectively (5′primer; 5′-ccgctcgagccaccatggcttccttggggcag-3′, 3′primer for B7-H4V; Similar primers for IgVC fragment of B7-H4 (5′ primer, 5′-cggaattccgctaatttatctctggcatact-3′ and 3′primer, 5′-cggaattccgctaagagttcagcaactgcag-3′) were also obtained. Upon amplification, PCR product was digested with XhoI and EcroRI and ligated into pcDNA3.1 vectors (Invitrogen, Carlsbad, CA). Purified plasmids were injected i.v. by hydrodynamic method. Briefly, 20 µg of plasmid DNA in 3 ml PBS was injected into the tail vein of B6 mice within 10 seconds one day before air pouches were generated as described in Supplemental Fig. 1. Gr-1+ neutrophils were quantified by flow cytometry. Each bar represents the means ± s.d. of six to eight mice in each group. *Significantly different from control vector, P<0.05.
- Figure S2. Decoy B7-H4 enhances neutrophil response to Listeria in B6 mice (JPG, 14.6 KB)
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Wild-type B6 mice of 6–8 week-old at groups of 5 were injected i.v. by hydrodynamic method as described in Fig. S1. with 30ug/2ml endotoxin-free plasmid DNA. One day later, mice were infected with 0.05 × 106 CFU of Listeria by i.v. injection. Mice were terminated two days post Listeria infection. Spleen Listeria load was analyzed by colony plating. Each bar represents the means ± s.d. of five mice in each group. *Significantly different from control vector, P<0.05.
- Figure S3. B7-H4KO mice have increased neutrophil response to LPS (JPG, 16.2 KB)
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The air pouch assay was performed as previously described (Edwards et al, J. Pathol. 134:147, 1981). Briefly, B7-H4KO or WT mice were anesthetized with 2, 2, 2-tribromoethanol (Sigma-Aldrich, St. Louis, Missouri) and subcutaneous dorsal pouches were created by injection of 5 ml of air. After 3 day, pouches were re-injected with 3 ml air. On day 6 after the first injection, 50 µg LPS in 1 ml PBS was injected into the pouches. Five hours later, Gr-1+ neutrophils were quantified by flow cytometry of cells rinsed from the pouch with sterile saline. Each bar represents the means ± s.d. of six to eight mice in each group. *Significantly different from WT, P<0.05.
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