Fig. 1. Loss of paracrine signaling in isolated single cells attenuates the LPS-stimulated secretion of some cytokines.

(A) LPS-induced inflammatory responses may be altered between cell populations (left) and isolated cells (right) because of the loss of paracrine signaling among isolated cells. (B) Intensities (arbitrary fluorescence units, AFU) of the indicated proteins secreted from single U937 cells treated with vehicle control (blue; n = 586 cells) or LPS for 20 hours (red; n = 601 cells) from one experiment that is representative of two or four independent experiments for control and LPS, respectively. The background threshold (BT) for each protein (black line) is calculated based on the zero-cell wells (see the Materials and Methods). The percentages of cells with secretion intensities above the BT are indicated. *P < 0.05 by Bonferroni-corrected Wilcoxon-Mann-Whitney test. (C) Comparison of the concentrations of the indicated secreted proteins in the culture medium of the cell population (plate) and the average concentration of all single cells cultured in the SCBC (Σ SC) for vehicle-treated (blue) and LPS-treated (red) cells. The protein concentrations in the culture medium of the cell population were measured by ELISA and are means ± standard error of the mean (SEM) of at least three biological replicates. Single-cell secretion intensities were converted to concentrations based on recombinant protein standard curves (fig. S3, and see the Materials and Methods), and concentrations for cells with intensities below the BT were set to zero. Values are means ± SEM of two (control) or four (LPS) biological replicates. **P < 0.05 by t test.