CD11c+ cells in Y-Ae-stained sections were demonstrated by first

CD11c+ cells in Y-Ae-stained sections were demonstrated by first staining with Y-Ae as described above, followed by additional H2O2/azide treatment and avidin and biotin blocking, to remove unreacted HRP and biotin/avidin, respectively. Sections were then incubated in either hamster anti-CD11c or hamster IgG (isotype control), biotinylated goat anti-hamster IgG, SA-HRP and Pacific Blue tyramide. Slides were mounted in Vectashield and images were captured using an Olympus BX-50 microscope with colour CCD digital camera and OpenLab digital imaging software (Improvision, Coventry, UK). In some images fluorochromes were false coloured to improve image

colour contrast. Results are expressed as mean ± SE mean when n ≥ 3 and mean ± range where n = 2. Student’s unpaired t tests with two-tailed distribution were used to calculate statistical significance (p < 0.05) when samples were normally distributed. Elegant Epacadostat studies by Itano et al. [1] described a novel system for studying Ag distribution, and identifying cells presenting Ag in vivo, in conjunction with Ag-specific CD4+ T cells recognising the same pMHC complex. We adapted these

tools to investigate Ag and APCs in the context of DNA vaccination. The original study [1] utilised an EαRFP (or EαDsRed) fusion protein for Ag detection. As others have reported cytotoxicity and aggregation Anti-diabetic Compound Library clinical trial associated with the DsRed1 protein used in this fusion protein and because we wanted to be able to further amplify the Ag signal, we developed an Ag detection system based on the monomeric eGFP. We modified the system described previously by replacing the RFP(DsRed1)-component

with a sequence aminophylline encoding eGFP and validated the EαGFP system for detection of both Ag and pMHC complexes in vivo. Subcutaneous immunisation with EαGFP protein resulted in marked heterogeneity in both Ag content and pMHC complex display in the cells of draining lymph nodes. Flow cytometric analysis of lymph node suspensions from mice immunised 24 h previously with 100 μg EαGFP protein plus 1 μg LPS showed that about 2.3–2.7% of all live cells were Y-Ae+ compared to about 0.4% for control mice immunised with LPS alone (Fig. 1A and B, upper panels). The Y-Ae isotype control antibody mIgG2b was used to set positive staining gates and showed approximately 0.2% background staining (Fig. 1A and B, lower panels). Hence, the maximum background Y-Ae staining (LPS and isotype control) is approximately 0.4% and staining above this level is considered positive staining. Background staining could not be completely eliminated due to tissue autofluorescence and the large numbers of cells that were acquired for analysis. The majority of Y-Ae+ cells found in draining lymph nodes at 24 h post-injection were GFPlow/− or below the level of GFP detection (∼2.0% of live cells, Fig. 1A, upper left quadrant) with only 0.

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