Figure 1.

Determination of the absolute cell number of circulating γδ T cells and their subsets in blood of PDAC patients. Fifty microliters whole blood samples from PDAC patients were stained with the indicated mAb in BD Trucount™ Tubes. These mAbs were previously titrated and a final concentration of 2–5 μg/ml was used. The mAb cocktail can be prepared in advance in bulk. The BD Trucount™ tubes contain lyophilized pellets that dissolve after adding liquid, thereby releasing a known number of fluorescent beads. Two hundred microliters of BD Lysing buffer was added to lyse red blood cells. To distinguish lymphocytes and beads from granulocytes and monocytes, an appropriate gate was set on CD45⁺ cells or beads using side scatter and CD45 or CD3 expression, respectively (upper panel). The ratio of the event number in the bead gate was compared to the total number of beads originally in the tube. The absolute cell number (Abs. Counts) of CD3⁺ (CD3), CD3⁺ TCRγδ⁺ (γδ), TCRγδ⁺ TCRnon-Vδ2⁺ (non-Vδ2), and TCRγδ⁺ TCRVδ2⁺ (Vδ2) within CD45⁺ lymphocytes was calculated as follows: (cells/microliter of whole blood) = [(events of cells of interest)/(ratio of acquired bead events to total beads in pellet)]/50 μl. Two representative determinations (PDAC-Donor 7 and 2) of 21 are shown, as are the percentages of the different cell populations.