Supplementary MaterialsSupplementary figures. a confocal microscope with CSU-W1 scanner unit MF-438 and CCD video camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung malignancy patients including an anti-PD-1 antibody treated individual were profiled of its GrB activity as proof of concept. Results: FGF9 GrB expression from your model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung malignancy patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade. Conclusion: As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a encouraging tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response. in vitroand in complex cell lysate. Quantification was carried out by measuring the switch in fluorescence as a result of the cleavage of a modular peptide by the said protease and the removal of a di-cysteine motif from peptide, which abrogates the bipartite tetracysteine display 24. Single-molecule detection technology have recently been reported MF-438 using F?rster resonance energy transfer (FRET) technology to count Cy5 bursts, which indicate the presence of target molecule 25. FRET altered substrates have also been developed MF-438 to accommodate different fluorescent pairs with unique excitation and emission wavelengths in order to obtain multiple signals of enzymes from single-cell encapsulated droplets and characterize protease activity profiles at single cell resolution 26. Being a common tool in clinical and biological labs and familiarity of most users, MF-438 fluorescence based detection was sought after in GrB measurement in this study. In this work, we fabricated a high throughput single cell screening microfluidic platform that can do compartmentalization and on-demand media exchange for repeated measurements. The current design of the microfluidic chip was inspired by the work of Armbrecht and Dittrich for parallel analysis and monitoring of a large number of isolated cells 12. Pneumatic valves were integrated into the chip to enable the quick and repeated fluid exchange. Cells were mechanically captured in hydrodynamic traps and isolated in individual microchambers of about 70 pL in volume with the actuation of the MF-438 pneumatic valves. A fluorometric activity assay was performed to measure GrB expression through its cleaving of a peptide substrate and release of AFC label. The expressed proteases from human immune cell lines (NK-92, GrB transduced Jurkat, and THP-1 cells) were compared using the single cell approach and the bulk approach. The platform was also applied to human PBMC samples from healthy donors and lung malignancy patients including anti-PD-1 antibody-treated patients. Cell surface marker staining was performed to distinguish specific cell populations generating GrB. Aside from GrB, immune cell expression of the other members of the Granzyme family can be investigated of their activities in immune response as well as collection of the cell of interest for further analysis as a possible extension of the study. Methods Microfluidic chip fabrication The microfluidic chip consists of two PDMS layers, one is a thin flow layer that contains an array of hydrodynamic traps as well as other microstructures that serve as filters, bubble traps, and pillar circulation guides. Here, cell samples and reagents were introduced through an inlet and made to flow through the channel with the use of a syringe pump (YSP-201, YMC Co. Ltd). The second is a thicker control layer with pneumatic valves that can be actuated with positive pressure to create a sealed chamber of.