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Near-IR fluorescence (NIRF) imaging is a new technology using IR fluorescent protein (iRFP) gene labelling and is potentially useful for in vivo applications. In the present study, we expressed iRFP and the TNF-related apoptosis inducing ligand gene in mesenchymal stem cells (MSCs) using adeno-associated virus (AAV) and showed that iRFP-labelled MSCs can be detected by fluorescence microscopy. We injected mice with MSCs labelled with AAV-iRFP, which we were then able to detect by whole-animal NIRF imaging. Our technique provides a visualizable, convenient and sensitive platform for research on tracking the fate of transplanted MSC cells in vivo.

The polystyrene bead-based flow cytometric immunoassay has been widely reported. However, the preparation of functional polystyrene bead is still inconvenient. This study describes a simple and easy on-bacterium flow cytometric immunoassay for protein quantification, in which Staphylococcus aureus (SAC) is used as an antibody-antigen carrier to replace the polystyrene bead. The SAC beads were prepared by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, paraformaldehyde fixation and antibody binding. Carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA 21-1) proteins were used as models in the test system. Using prepared SAC beads, biotinylated proteins, and streptavidin-phycoerythrin (SA-PE), the on-bacterium flow cytometric immunoassay was validated by quantifying CEA and CYFRA 21-1 in sample. Obtained data demonstrated a concordant result between the logarithm of the protein concentration and the logarithm of the PE mean fluorescence intensity (MFI). The limit of detection (LOD) in this immunoassay was at least 0.25 ng/ml. Precision and accuracy assessments appeared that either the relative standard deviation (R.S.D.) or the relative error (R.E.) was

This study investigated a cytometric dual-bead array used to analyze PML-RARalpha fusion protein. The carboxylated and aminated polystyrene beads were prepared and barcoded with either high or low brightness using a fluorescein isothiocyanate (FITC) penetration method. Using anti-RARalpha antibody, homotype control antibody, barcoded beads and phycoerythrin (PE)-labeled antibody, the analytical capability of the cytometric dual-bead array was tested with two cell models containing a NB4 cell line. Fusion protein levels were normalized using a PE mean fluorescence intensity (MFI) ratio (PE MFI of high brightness beads/PE MFI of low brightness beads). In flow cytometry, the cytometric dual-bead array could analyze PML(L)-RARalpha protein with high specificity, and this assay possessed analytical sensitivity of at least 0.6%. A dilution experiment also demonstrated a concordant result between the logarithm of the PE MFI ratio and the logarithm of the NB4 cell concentration (R2 = 0.9936). When compared with a cytometric single-bead assay, this array exhibited a lower relative standard deviation (R.S.D.) and a lower relative error (R.E.). In conclusion, using the MFI ratio can attenuate data error in fusion protein cytometric analysis, and the cytometric dual-bead array presented here may be of use in the quantification of PML-RARalpha fusion protein.