Incubation time was chosen on the basis of CTC reduction kinetics in cells that indicated that a pseudo-plateau had been reached at that time. The same series were prepared with cells taken out of the incubator and left at room temperature for 2 h before the test and with cells previously fixed in 3.7% formaldehyde solution in PBS for 1 h at room temperature. FCM dot plots and histograms of GFP, CTC and Sytox red fluorescence intensities collected in their Resiquimod respective emission channels, i.e. FL1, FL3 and FL4, versus forward scattering, are shown in Fig. 1. Subcellular debris was removed from the analysis by gating the data to an SSC+ region, corresponding to SSC values higher than 10. Then, we defined for each fluorescence channel a threshold value delimiting positive and negative regions with respect to the considered marker and we determined the corresponding FCM parameters, cell percentage and fluorescence intensity. Our results showed that no significant difference in GFP expression or Sytox red labelling was observed between fresh and bench cells. In contrast, both cell distribution between CTC + and CTC- regions and mean fluorescence intensity were noticeably affected in bench cells compared with fresh cells, indicating a drop in the reduced CTC production consistent with a cell respiring R 568 hydrochloride activity decline caused by a temperature and oxygenation decrease. In the extreme situation of the fixed cells, CTC reduction was abolished. Concomitantly, more than 95% of cells exhibited Sytox red labelling, and partial loss of GFP was observed. These results showed that CTC reduction sharply discriminated live from dead cells, as did Sytox red, but also measured graded respiration levels of healthy cells in which GFP content was unchanged on a 2 h time scale. Therefore, GFP intensity could be used as an internal standard to normalize reduced CTC production and define a single-cell respiration index, fR= flCTC/flGFP. To check that this normalization introduced no spreading or deformation of the value distribution compared to raw flCTC, we compared the distributions of fR and flCTC values normalized to the mean. Thus, our results indicate that bacteria detected the formation of cell surface contact and responded to it by metabolism downmodulation, as confirmed by the decrease in respiring activity. At this stage, we sought to further analyze the respiration activity decrease observed in particle-attached cells so as to determine whether the observed cell response actually resulted from cell-artificial surface anchorage or from cell-cell associations occurring on the particle surface. The results shown above thus indicated that respiring activity decreased upon single cell-cell or cell-substrate contact at the surface of a bacterium after 40 min.