Schematic shows the principles of a collapsible roof device. (a) Fabrication of CRD is achieved through reversible assembly of PDMS layers held to the substrate by vacuum. The top layer with tubing connections, the middle membrane layer with an array of individual microchambers as the collapsible roof of the microfluidic channel, and the bottom layer with channels for sample liquid and vacuum network. (b) A diagram of the CRD on an Ab printed hydrogel substrate with its microchambers aligned to the Ab microarrays. Each microchamber has 2 columns of cell capturing Ab spots and 2 columns of anti-cytokine capturing Ab spots. (c) CRD operation process begins with (i) cell seeding and stimulation, (ii) inlet of microfluidic channel is clamped while the sample continues to be drawn out from the outlet, (iii) the roof of the channel is brought down and the outlet is clamped.
The photographs show the CRD loaded with red dye in the (a) open and (b) collapsed configurations. A clear volume change between the two states illustrates isolation and localization of working samples within individual microchambers.
Immunofluorescent staining of cells captured on adjacent anti-CD4 and anti-CD8 microarrays. The top row cells are CD8+ cells and the bottom row cells are CD4+ cells. (a) Fluorescent image of CD4+ and CD8+ cells stained with anti-CD3-FITC (green). (b) Fluorescent image of CD4+ and CD8+ cells stained with anti-CD8-PE (red). (c) Merged fluorescent image of the two images showing both CD4 and CD8 cells were stained for anti-CD3-FITC (green) while only CD8 cells were stained for anti-CD8-PE (red) thus indicating a high purity and specificity of cell capture.
Merged brightfield and fluorescent images of (a) sample incubated in the open CRD under mitogenic stimulation for 5 h and (b) sample incubated in the collapsed CRD under mitogenic stimulation for 5 h. (c) Relative fluorescent intensity values were normalized based on a per cell basis and graphed to compare the signal advantage in cytokine capture that the collapsed configuration has over the open configuration.
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