Preparation of Functionalized Magnetic Nanoparticles for Time-Resolved Fluorescent Imaging

To begin, add streptavidin functionalized nanoparticles to tubes for TNF alpha, interleukin-1 beta, and interleukin-6 detection. Dilute the streptavidin plus nanoparticles in equal volumes of PBS. Then, add the biotinylated capture antibody solutions to tubes and incubate for 30 minutes at room temperature.

Next, add 0.01%of 1 millimolar D-Biotin solution to each tube and incubate for five minutes. Then, hold a neodymium magnet close to the tube to collect the particles. Discard the clear supernatant and immediately resuspend the nanoparticles in a mixture of Pluronic F-127 and PBS and incubate for 30 minutes.

After incubating the particles for 30 minutes in storage buffer, stir them to ensure a uniform mixture. Immediately before encapsulation, combine the conjugated nanoparticles for interleukin-6, TNF alpha, and interleukin-1 beta. Now, wash the particles with complete media using the magnet as demonstrated earlier, and resuspend the beads in complete media.

Afterward, add detection antibodies for interleukin-6, TNF alpha, and interleukin-1 beta to achieve the final concentration of 10 nanomolar.