Journal: PLoS ONE
Article Title: Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status
Figure Lengend Snippet: FISH-Flow detection of antigen-specific T cell responses associated with latent tuberculosis infection. PBMCs from 65 LTBI+ and LTBI- donors were cultured +/- PPD stimulation, stained with FITC-labeled αCD3 antibody, hybridized with Cy5-labeled nucleic acid probes specific for IFNG, IL2, and no target control (GFP) and analyzed by flow cytometry, as detailed in the legend to Fig 1 . (a) Representative contour plots showing results with cells from an LTBI+ (top row) and an LTBI- (bottom row) donor, unstimulated and PPD-stimulated, as indicated. The rightmost panels in both rows show IFNG induction with PMA/Ionomycin stimulation as positive control. The upper right quadrant of each plot shows the frequencies of mRNA+CD3+ cells. (b) Frequencies of mRNA+CD3+ cells in the 65-donor population, by LTBI and stimulation status. The box plots show lower quartile, median, and upper quartile of the distribution. The lower whisker is the minimum, while the upper limit of the whisker represents the median + 1.5 x the interquartile range (values exceeding the upper limit of the whisker are shown as circles). Additionally, the mean is shown as (+) symbol. To confirm the reproducibility of the assay, selected donors were assayed twice with blood samples drawn > 4 months apart.
Article Snippet: PBMC collection and cell culture Within two hours after blood collection, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare, Waukesha, WI) density-gradient centrifugation and stored in liquid nitrogen.
Techniques: Fluorescence In Situ Hybridization, Flow Cytometry, Infection, Cell Culture, Staining, Labeling, Cytometry, Positive Control, Whisker Assay