Forkhead transcription factors play critical roles in leukocyte homeostasis. To study further the immunological functions of Foxo1, we generated mice that selectively lack Foxo1 in T cells (Foxo1 flox/flox Lck.cre 1 conditional knockout mice (cKO)). Although thymocyte development appeared relatively normal, Foxo1 cKO mice harbored significantly increased percentages of mature single positive T cells in the thymus as compared with WT mice, yet possessed smaller lymph nodes and spleens that contained fewer T cells. Foxo1 cKO T cells were not more prone to apoptosis, but instead were characterized by a CD62L lo CCR7 lo CD44 hi surface phenotype, a poorly populated lymphoid compartment in the periphery, and were relatively refractory to TCR stimulation, all of which were associated with reduced expression of Sell, Klf2, Ccr7, and S1pr1. Thus, Foxo1 is critical for naïve T cells to populate the peripheral lymphoid organs by coordinating a molecular program that maintains homeostasis and regulates trafficking.
Acoustic droplet ejection (ADE) enables crystallization experiments at the low-nanoliter scale, resulting in rapid vapor diffusion equilibration dynamics and efficient reagent usage in the empirical discovery of structure-enabling protein crystallization conditions. We extend our validation of this technology applied to the diverse physicochemical property space of aqueous crystallization reagents where dynamic fluid analysis coupled to ADE aids in accurate and precise dispensations. Addition of crystallization seed stocks, chemical additives, or small-molecule ligands effectively modulates crystallization, and we here provide examples in optimization of crystal morphology and diffraction quality by the acoustic delivery of ultra-small volumes of these cofactors. Additional applications are discussed, including set up of in situ proteolysis and alternate geometries of crystallization that leverage the small scale afforded by acoustic delivery. Finally, we describe parameters of a system of automation in which the acoustic liquid handler is integrated with a robotic arm, plate centrifuge, peeler, sealer, and stacks, which allows unattended high-throughput crystallization experimentation.
Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio.
Recently identified associations between variants of cancer genes and drug resistance have increased the value for comprehensive drug sensitivity screening in combination with molecular profiling of cancer cells. This in turn, has led to a demand for improvements to screening throughput and efficiency. Echo® Liquid Handlers use acoustic energy to provide high throughput, non-contact, liquid handling for a range of applications. Non-contact transfer avoids the risk of cross-contamination, eliminates tip costs, and facilitates the progression to high density assay formats. Echo Liquid handlers precisely and accurately transfer 2.5 nL droplets of sample and reagent, which enables the miniaturization of biochemical screens. This poster discusses the implementation of miniaturized drug sensitivity screening, at the Institute of Molecular Medicine in Finland (FIMM), with assay-ready plates produced by the Echo liquid handler. Citation Format: Bonnie Edwards. The advantages of acoustic liquid handling for drug sensitivity screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5393. doi:10.1158/1538-7445.AM2014-5393
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