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. 2010 Feb 10;28(6):1620–1634. doi: 10.1016/j.vaccine.2009.11.014

Fig. 5.

Fig. 5

pDNA distribution and phenotype of pDNA-containing cells following plasmid injection. (A) Tissues including the TA muscle, draining popliteal (popLN) and inguinal lymph nodes (ILN), distal brachial and cervical lymph nodes (distal LNs), spleen, peripheral blood and bone marrow were collected 1 h and 24 h following intramuscular injection of Cy5-labelled (pDNA-Cy5) or unlabelled control pDNA. Cell suspensions were examined for the presence pDNA-Cy5 by flow cytometry. Live cell gates (determined by forward and side scatter characteristics) were drawn and cell suspensions from mice receiving unlabelled pDNA were used to set cutoffs for positive and negative gates and represented background tissue staining. Cy5 fluorescence was assessed using the FL4 channel of the FACScalibur and cells with above background fluorescence were considered positive. (A) Cy5 fluorescence (X-axis) vs. side scatter (Y-axis) for representative samples from various tissues at 2 different timepoints. At 1 h we observed pDNA-Cy5 in popliteal, inguinal and distal peripheral lymph nodes with largest numbers in the local muscle-draining popliteal LN. We also observed a few pDNA-Cy5+ cells in peripheral blood, but none were detected in either spleen or bone marrow. Asterisk (*) indicates tissues with Cy5+ cells. At 24 h post-pDNA-Cy5 injection we found Cy5+-cells in draining and distal peripheral lymph nodes. The popliteal LN contained the highest percentage of positive cells. Although we were unable to find pDNA-containing cells in the peripheral blood at 24 h, we were able to demonstrate positive cells in both the spleen and BM. Further characterisation of cell suspensions from pDNA-Cy5 immunised mice shown that pDNA-Cy5+ cells from the popLN and spleen were MHCII+, whereas those from BM were predominantly MHCII− (B). Statistical comparisons (Students’ unpaired t-test, 2 way, assuming unequal variance) were made between the percentage of Cy5+-live cells in pDNA-Cy5 injected mice and background staining from unlabelled mice. (C) The mean percentage of Cy5+ cells in popLN, spleen and BM. Asterisk (*) indicates statistical significance where p < 0.05 (n = 3).