To begin, add freshly prepared 4.5 milliliters of EDC working solution to a 15 milliliter polypropylene tube containing the amine-functionalized polystyrene beads and TCPP in MES buffer. Place the tube in an inverting rotator at 35 RPM for 16 hours at room temperature protected from light. Then, centrifuge it for 10 minutes.
Aspirate the supernatant and re-suspend the beads in 0.8 milliliters of Hank's Balanced Salt Solution. Then, using a one milliliter pipette, transfer the bead solution to an amber polypropylene vial and store it at four degrees Celsius. The relationship between the particulate formation, the median, fluorescence intensity or MFI, and the bead TCPP/EDC ratio showed that with increasing ratios, the MFI increased until it formed a plateau.
The particulates appeared only when EDC was used as a coupling reagent during the labeling procedure. Labeling the beads with TCPP alone resulted in much lower fluorescence intensity. When TCPP-labeled beads were tested after 300 days, no significant change in the MFI or in the particulate content was observed.
The coupling of TCPP to the beads had a minimum effect on its fluorescence emission spectrum. However, its spectrum differed more from TCPP-labeled A549 lung cancer cells. In a multi-flora four labeled sputum sample, the TCPP-labeled beads worked equally well as the TCPP-stained A549 cells.
