shaking (around 20 hours). 26106 yeast induced cells were washed with 1 ml of washing buffer (PBS, BSA 0.5%, EDTA 20mM) and resuspended in 100 ml containing: i) 1 ml of anti-SV5 mAb (1 mg/ ml) plus 100 nM biotinylated antigen together; ii) anti-SV5 alone; or iii) biotinylated antigen alone. After 30 minutes of incubation at RT followed by 5 minutes on ice, cells were spun down and washed twice with 500 ml of ice-cold washing buffer. Cells were resuspended in 100 ml of a 1/200 dilution of secondary reagents: streptavidin-Alexa633 for antigen detection and anti-mouse-PE conjugated for anti SV5-scFv detection. The cells were incubated for 30 minutes in ice, harvested and washed twice with cold buffer. Cells were then resuspended in 1 ml of PBS or GFP1-10 solution (3.75 mg/ml, 4 mM). Florescence data were collected using BD FACSAria flow Cytometer and analyzed using DIVA software. The yeast population was gated and the mean fluorescent values for PE (corresponding to scFv expression), APC (corresponding to antigen binding) and FITC (corresponding to complementation of scFv- D1.3-GFP11 with GFP10) were recorded using appropriate excitation lasers and emission filters. Split GFP Labeling of scFvs Multiplexed Flow Cytometry Three different color-coded, carboxylated microsphere sets were used (Luminex, xMAP 135, 129, 159). Each individual set was coupled with Neutravidin, coupling was quantified [24], and biotinylated antigens were respectively bound: lysozyme to beads 135, myoglobin to beads 129 and IgE receptor to beads 159. 16 ml (1.66105 microspheres) of each bead set were incubated with 100 ml of different concentrations of purified protein (ranging from 0.38 to 380 pmol) or with POP culture extract. The solutions were then incubated with a molar excess of GFP1-10 for two hours at RT, and the mean fluorescence data of each bead set was collected using the high-throughput analysis feature of a Becton Dickinson LSRII Flow Cytometer and analyzed using the DIVA software package. The bead multiplex was separated into gates using the setting previously described [24] and the mean fluorescent value of each gate was recorded following excitation using 488 nm laser through FITC detectors. Equilibrium binding titration curves to determine the affinity constant Kd of scFv-D1.3- GFP11 was obtained by nonlinear regression using PRISM software (version 5.0 Graph Pad). Figure 3. Progress curves analysis of the complemented fluorescence. A. Progress curves of complemented fluorescence using purified protein. The increasing fluorescence over time was determined (a.u arbitrary units) for the complementation of scFv-D1.3-GFP11 samples at different concentrations (300, 150, 75 pmol) incubated with an excess of GFP1-10 (800 pmol). Fluorescence (lexc = 488 nm/lem = 530 nm) was monitored at 3 minutes intervals for 20 h at RT. B. Progress curves of complemented fluorescence using serial dilutions of crude bacteria extract. A similar trend of increasing fluorescence was measured (a.u arbitrary units) directly using serial dilution of crude bacterial culture extract, albeit at lower fluorescent values. Fluorescence (lexc = 488 nm/lem = 530 nm) was monitored at 3 minutes intervals for 10 hours at RT. doi:10.1371/journal.pone.0025727.g003 PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e25727