Guava expressplus

The BacLight Bacterial Membrane Potential Kit (Cat # B34950, Molecular Probes, Inc., Life Technologies Corporation, Grand Island, NY) was used for flow cytometric measurement of membrane potential of untreated versus plasma treated bioaerosls. A Guava Flow Cytometer and Guava ExpressPlus assay software (EMD Millipore, Billerica, MA) were employed for the assay, following the direction of manufacturer. The assay employ solution of carbocyanine dye DiOC₂(3) (3,3’-diehtyloxa-carbocyanine iodide). DiOC₂(3) exhibits green fluorescence in all bacteria, but the fluorescence shifts toward red emission as the dye molecules self-associate at the higher cytosolic concentrations caused by larger membrane potential. Therefore a fluorescence ratio (red/green) is calculated to demonstrate any depolarization of membrane by comparing experimental conditions versus control (untreated) cell samples. The following formula was used to determine the number of bacteria in the sample:
$\text{Bacteria}/\text{mL}=\left(\left(number of events in bacteria region\right)\times \left(dilution factor\right)\right)/\left(number of events in bead region\right)\times {10}^{-6})$
Finally, the fluorescence ratios (red/green) for various treatment conditions were graphed.

Cell-surface analysis of hBFP-ASCs was by flow cytometry [14 (link)]. Fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibodies against CD14, CD90 and CD105 (Abcam, Cambridge, MA, USA) and CD34 and CD44 (Becton & Dickinson, San Jose, CA, USA) were used. Phycoerythrin-conjugated mouse IgG1 (Becton & Dickinson) was used as a negative control. Data acquisition and analyses were performed with Guava Express Plus (version 5.3) software (EMD Millipore Corporation, Billerica, MA, USA).