F. Cytokine detection using the bead-based magnetic immune assay

Four cytokines, namely, TNFα, IL-1β, IL-6, and IL-10, were quantified using a magnetic bead-based immune assay by employing the Milliplex Map, Human Cytokine magnetic bead panel, Cat # HCYOMAG-60k (Millipore, USA). The device structure and the bead-based agitation enable continuous transport of a homogeneous cell culture supernatant from the cell culture chamber into the five immune assay channels through the porous membrane. Each channel contains magnetic beads coated with a biotinylated cytokine capture antibodies against the cytokine of interest. Serial dilutions of the cytokines standards with known concentrations were prepared, and the cytokine detection was conducted in a 96-well plate according to the manufacturer's instruction. However, the fluorescent signals were measured using a fluorescent plate reader instead of using the Luminex system without detecting the bead color. The number of inoculated THP-1 cells in the cell chamber was within the range of 3–5 × 104 cells. After cell inoculation, the inlet and outlet of the cell chamber were blocked to prevent cell loss and to maintain uniform flow within the cell culture chamber with low mechanical shear stress. The cells were supplied with the culture media through the upper chamber where it is perfused through the BM membrane. One fluidic port of the perfusion chamber was connected to a 50 ml media reservoir, and the second port was connected to a 10 ml plastic syringe, which is mounted on a syringe pump (PHD Ultra syringe pumps, Harvard Apparatus, USA). The culture media reservoir was kept together with the chip inside the CO2 incubator, and the syringe pump (outside the incubator) was operated in the withdrawal mode. Five different magnetic bead suspensions, corresponding to the cytokine of interest, with a volume of 20 μl and a concentration of 2 × 106 beads/ml, with a total number of beads within each channel of 4 × 105, were incubated in the five immune assay channels, while the culture media with/without stimuli are perfused [Fig. 2(f)]. Then, the bead suspensions were collected after various incubation periods, specifically after 2, 6, 24, 24, 36, and 72 h of incubation. The beads within the immune channels are retained while agitated, thanks to the magnetic field generated by the MAS. The continuous agitation enhances the chance of bead–cytokine conjugation. The culture media (with/without) stimuli follow the following route: entering the perfusion chamber through the perfusion inlet → passing through the BM membrane into the cell chamber → passing through the porous membrane into the bead channels. During the perfusion or incubation, the MAS is kept under the chip while it is in the operation mode. To collect the beads at the end of the incubation period, the MAS is shifted away from the chip and 30 μl of fresh media (without stimuli) was injected into each channel [Fig. 2(h)]. Then, fresh magnetic bead suspensions (each with a volume of 20 μl) were -injected into the channels for the next incubation. The collected biotinylated beads were washed three times with PBS. Then, streptavidin–PE conjugated cytokine detection antibodies were added to the bead suspensions, and the samples were incubated for 2 h. Then, the bead–cytokine–streptavidin–PE complexes were washed with PBS, and the fluorescent signal was measured using the fluorescent plate reader.