Stanford Advisors

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All Publications

  • Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery. Lab on a chip Brower, K. K., Carswell-Crumpton, C., Klemm, S., Cruz, B., Kim, G., Calhoun, S. G., Nichols, L., Fordyce, P. M. 2020


    Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate individual variants-of-interest at high-throughput remains challenging. Here, we present sdDE-FACS (s[combining low line]ingle d[combining low line]roplet D[combining low line]ouble E[combining low line]mulsion-FACS), a new method that uses a standard flow cytometer to phenotype, select, and isolate individual double emulsion droplets of interest. Using a 130 mum nozzle at high sort frequency (12-14 kHz), we demonstrate detection of droplet fluorescence signals with a dynamic range spanning 5 orders of magnitude and robust post-sort recovery of intact double emulsion (DE) droplets using 2 commercially-available FACS instruments. We report the first demonstration of single double emulsion droplet isolation with post-sort recovery efficiencies >70%, equivalent to the capabilities of single-cell FACS. Finally, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets via qPCR with little to no cross-contamination. sdDE-FACS marries the full power of droplet microfluidics with flow cytometry to enable a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis.

    View details for DOI 10.1039/d0lc00261e

    View details for PubMedID 32417874

  • Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads. eLife Nguyen, H. Q., Roy, J., Harink, B., Damle, N. P., Latorraca, N. R., Baxter, B. C., Brower, K., Longwell, S. A., Kortemme, T., Thorn, K. S., Cyert, M. S., Fordyce, P. M. 2019; 8


    Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of human calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.

    View details for DOI 10.7554/eLife.40499

    View details for PubMedID 31282865

  • Live imaging of Aiptasia larvae, a model system for coral and anemone bleaching, using a simple microfluidic device. Scientific reports Van Treuren, W., Brower, K. K., Labanieh, L., Hunt, D., Lensch, S., Cruz, B., Cartwright, H. N., Tran, C., Fordyce, P. M. 2019; 9 (1): 9275


    Coral reefs, and their associated diverse ecosystems, are of enormous ecological importance. In recent years, coral health has been severely impacted by environmental stressors brought on by human activity and climate change, threatening the extinction of several major reef ecosystems. Reef damage is mediated by a process called 'coral bleaching' where corals, sea anemones, and other cnidarians lose their photosynthetic algal symbionts (family Symbiodiniaceae) upon stress induction, resulting in drastically decreased host energy harvest and, ultimately, coral death. The mechanism by which this critical cnidarian-algal symbiosis is lost remains poorly understood. The larvae of the sea anemone, Exaiptasia pallida (commonly referred to as 'Aiptasia') are an attractive model organism to study this process, but they are large (100 mm in length, 75 mm in diameter), deformable, and highly motile, complicating long-term imaging and limiting study of this critical endosymbiotic relationship in live organisms. Here, we report 'Traptasia', a simple microfluidic device with multiple traps designed to isolate and image individual, live larvae of Aiptasia and their algal symbionts over extended time courses. Using a trap design parameterized via fluid flow simulations and polymer bead loading tests, we trapped Aiptasia larvae containing algal symbionts and demonstrated stable imaging for >10 hours. We visualized algae within Aiptasia larvae and observed algal expulsion under an environmental stressor. To our knowledge, this device is the first to enable time-lapsed, high-throughput live imaging of cnidarian larvae and their algal symbionts and, in further implementation, could provide important insights into the cellular mechanisms of cnidarian bleaching under different environmental stressors. The 'Traptasia' device is simple to use, requires minimal external equipment and no specialized training to operate, and can easily be adapted using the trap optimization data presented here to study a variety of large, motile organisms.

    View details for DOI 10.1038/s41598-019-45167-2

    View details for PubMedID 31239506

  • High-throughput chromatin accessibility profiling at single-cell resolution. Nature communications Mezger, A., Klemm, S., Mann, I., Brower, K., Mir, A., Bostick, M., Farmer, A., Fordyce, P., Linnarsson, S., Greenleaf, W. 2018; 9 (1): 3647


    Here we develop a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access to DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across a massively parallel (5184) nano-well array, yielding a nearly 20-fold improvement in throughput (up to ~1800 cells/chip, 4-5h on-chip processing time) and library preparationcost (~81 per cell) compared to prior microfluidic implementations. We apply this method to measure regulatory variation in peripheral blood mononuclear cells (PBMCs) and show robust, de novo clustering of single cells by hematopoietic cell type.

    View details for PubMedID 30194434

  • An Open-Source, Programmable Pneumatic Setup for Operation and Automated Control of Single- and Multi-Layer Microfluidic Devices. HardwareX Brower, K., Puccinelli, R., Markin, C. J., Shimko, T. C., Longwell, S. A., Cruz, B., Gomez-Sjoberg, R., Fordyce, P. M. 2018; 3: 117–34


    Microfluidic technologies have been used across diverse disciplines (e.g. high-throughput biological measurement, fluid physics, laboratory fluid manipulation) but widespread adoption has been limited in part due to the lack of openly disseminated resources that enable non-specialist labs to make and operate their own devices. Here, we report the open-source build of a pneumatic setup capable of operating both single and multilayer (Quake-style) microfluidic devices with programmable scripting automation. This setup can operate both simple and complex devices with 48 device valve control inputs and 18 sample inputs, with modular design for easy expansion, at a fraction of the cost of similar commercial solutions. We present a detailed step-by-step guide to building the pneumatic instrumentation, as well as instructions for custom device operation using our software, Geppetto, through an easy-to-use GUI for live on-chip valve actuation and a scripting system for experiment automation. We show robust valve actuation with near real-time software feedback and demonstrate use of the setup for high-throughput biochemical measurements on-chip. This open-source setup will enable specialists and novices alike to run microfluidic devices easily in their own laboratories.

    View details for PubMedID 30221210

  • Programmable Microfluidic Synthesis of Over One Thousand Uniquely Identifiable Spectral Codes ADVANCED OPTICAL MATERIALS Nguyen, H. Q., Baxter, B. C., Brower, K., Diaz-Botia, C. A., DeRisi, J. L., Fordyce, P. M., Thorn, K. S. 2017; 5 (3)
  • Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices. Journal of visualized experiments : JoVE Brower, K., White, A. K., Fordyce, P. M. 2017


    Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

    View details for DOI 10.3791/55276

    View details for PubMedID 28190039

  • Peptide library synthesis on spectrally encoded beads for multiplexed protein/peptide bioassays Nguyen, H. Q., Brower, K., Harink, B., Baxter, B., Thorn, K. S., Fordyce, P. M., Gray, B. L., Becker, H. SPIE-INT SOC OPTICAL ENGINEERING. 2017

    View details for DOI 10.1117/12.2254908

    View details for Web of Science ID 000405953000023

  • Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lu, Y., Xue, Q., Eisele, M. R., Sulistijo, E. S., Brower, K., Han, L., Amir, E., Pe'er, D., Miller-Jensen, K., Fan, R. 2015; 112 (7): E607–E615


    Despite recent advances in single-cell genomic, transcriptional, and mass-cytometric profiling, it remains a challenge to collect highly multiplexed measurements of secreted proteins from single cells for comprehensive analysis of functional states. Herein, we combine spatial and spectral encoding with polydimethylsiloxane (PDMS) microchambers for codetection of 42 immune effector proteins secreted from single cells, representing the highest multiplexing recorded to date for a single-cell secretion assay. Using this platform to profile differentiated macrophages stimulated with lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4), reveals previously unobserved deep functional heterogeneity and varying levels of pathogenic activation. Uniquely protein profiling on the same single cells before and after LPS stimulation identified a role for macrophage inhibitory factor (MIF) to potentiate the activation of LPS-induced cytokine production. Advanced clustering analysis identified functional subsets including quiescent, polyfunctional fully activated, partially activated populations with different cytokine profiles. This population architecture is conserved throughout the cell activation process and prevails as it is extended to other TLR ligands and to primary macrophages derived from a healthy donor. This work demonstrates that the phenotypically similar cell population still exhibits a large degree of intrinsic heterogeneity at the functional and cell behavior level. This technology enables full-spectrum dissection of immune functional states in response to pathogenic or environmental stimulation, and opens opportunities to quantify deep functional heterogeneity for more comprehensive and accurate immune monitoring.

    View details for DOI 10.1073/pnas.1416756112

    View details for Web of Science ID 000349446000003

    View details for PubMedID 25646488

    View details for PubMedCentralID PMC4343126

  • Limit of detection of field effect transistor biosensors: Effects of surface modification and size dependence APPLIED PHYSICS LETTERS Rajan, N. K., Brower, K., Duan, X., Reed, M. A. 2014; 104 (8)

    View details for DOI 10.1063/1.4867025

    View details for Web of Science ID 000332619100154

  • A microchip platform for interrogating tumor-macrophage paracrine signaling at the single-cell level LAB ON A CHIP Elitas, M., Brower, K., Lu, Y., Chen, J. J., Fan, R. 2014; 14 (18): 3582–88


    It is increasingly recognized that infiltrating immune cells contribute to the pathogenesis of a wide range of solid tumors. The paracrine signaling between the tumor and the immune cells alters the functional state of individual tumor cells and, correspondingly, the anticipated response to radiation or chemotherapies, which is of great importance to clinical oncology. Here we present a high-density microchip platform capable of measuring a panel of paracrine signals associated with heterotypic tumor-immune cell interactions in the single-cell, pair-wise manner. The device features a high-content cell capture array of 5000+ sub-nanoliter microchambers for the isolation of single and multi-cell combinations and a multi-plex antibody "barcode" array for multiplexed protein secretion analysis from each microchamber. In this work, we measured a panel of 16 proteins produced from individual glioma cells, individual macrophage cells and varying heterotypic multi-cell combinations of both on the same device. The results show changes of tumor cell functional phenotypes that cannot be explained by an additive effect from isolated single cells and, presumably, can be attributed to the paracrine signaling between macrophage and glioma cells. The protein correlation analysis reveals the key signaling nodes altered by tumor-macrophage communication. This platform enables the novel pair-wise interrogation of heterotypic cell-cell paracrine signaling at the individual cell level with an in-depth analysis of the changing functional phenotypes for different co-culture cell combinations.

    View details for DOI 10.1039/c4lc00676c

    View details for Web of Science ID 000340474300019

    View details for PubMedID 25057779

    View details for PubMedCentralID PMC4145007

  • Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction. Frontiers in oncology Kwak, M., Mu, L., Lu, Y., Chen, J. J., Brower, K., Fan, R. 2013; 3: 10-?


    Secreted proteins including cytokines, chemokines, and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g., in the immunological system (Rothenberg, 2007), tumor microenvironment (Hanahan and Weinberg, 2011), or stem cell niche (Gnecchi etal., 2008). Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically identical cell population can give rise to diverse phenotypic differences (Niepel etal., 2009). Non-genetic heterogeneity is also emerging as a potential barrier to accurate monitoring of cellular immunity and effective pharmacological therapies (Cohen etal., 2008; Gascoigne and Taylor, 2008), but can hardly assessed using conventional approaches that do not examine cellular phenotype at the functional level. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

    View details for DOI 10.3389/fonc.2013.00010

    View details for PubMedID 23390614

    View details for PubMedCentralID PMC3565185

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