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Dead cells were defined as events in the Prochlorococcus gate (forward angle light scatter and red fluorescence) that exhibited green fluorescence values similar to a glutaraldehyde-killed control.
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Doublets were excluded based on a gate on forward height versus area, and viable lymphocytes were identified using a gate based on forward and side scatter, and by using the Live/Dead staining reagent.
Cells were initially gated to remove doublets, followed by a lymphocytes gate on forward scatter area versus side scatter area.
For the subsequent analysis, only those cells in the 'live' gate by forward and side scatter were used.
Cells were restricted to a live-cell gate by forward and side-scatter parameters, and cell populations of interest were captured by standardized polygonal gates.
Cells were gated in forward scatter/side scatter, and the median of the gated fluorescence peak was used.
In each set, live cells were gated using forward scatter channel vs side scatter channel followed by gating on the IDO-expressing mCherry-positive cells.
Live PBMCs were electronically gated by forward- and right-angle scatters; different types of cells were then gated and the percentage and intensity of expression were evaluated.
To avoid nonspecific fluorescence from dead cells, live cells were gated using forward and side scatter.
CD3+ lymphocytes were gated using forward scatter, side scatter, and FITC channels.
Viable cells were gated using forward and side scatter characteristics.
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