Isolation of epithelial cells and leukocytes from intestinal tissues with DTT, EDTA and collagenase treatments.

<p>Representative dot plots of total cells from (<b>A</b>) DTT wash (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IV); (<b>B</b>) lower layer of percoll density gradient, isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IX); (<b>C</b>) lower layers of percoll density gradient, isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step XIII) from jejunum tissue showing distribution of epithelial cells (ECs; cytokeratin as an epithelial cell marker) and CD45 leukocytes in a normal uninfected healthy rhesus macaque. Each quadrant shows percentages of specified cell populations. Note increased percentage of ECs were isolated from DTT wash compared to other methods examined. Mean frequencies of cytokeratin (CK) positive, CD45 positive, double positive and double negative for CK and CD45 cell subsets are shown as box and whisker vertical bars for different isolation protocols as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>. In summary all the specified bars represent (<b>D</b>) cells isolated from DTT wash (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IV); (<b>E</b>) upper layer cells from percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IX); (<b>F</b>) lower layer cells from percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IX); (<b>G</b>) upper layer cells from percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step XIII); and (<b>H</b>) lower layer cells from percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step XIII) from jejunum in healthy, normal, uninfected rhesus macaques (n = 5). Note that in all isolation protocols, there was a variable amount of CD45+ and double negative CK−CD45− cells contamination observed. * Indicates significant differences between CK+CD45− and other different subsets of total cells within the specified isolation protocol.