Supplementary Material, Perez_supplemental_Material_final – High-Throughput Flow Cytometry Drug Combination Discovery with Novel Synergy Analysis Software, SynScreen

<p>Supplementary Material, Perez_supplemental_Material_final for High-Throughput Flow Cytometry Drug Combination Discovery with Novel Synergy Analysis Software, SynScreen by Dominique R. Perez, Bruce S. Edwards, Larry A. Sklar, and Alexandre Chigaev in SLAS Discovery</p

Secondary screening of neuroprotective compounds.

<p>Compounds identified through high-throughput screening were administered at 0.1 (white), 1 (light gray), 10 (dark gray), and 100 µM (black) at reoxygenation after 2 hours OGD. Twenty-four hours later, cells survival was measured by TUNEL assay and compared to DMSO vehicle-treated neurons. Compounds providing at least 2-fold increased neuroprotection over vehicle were further investigated. MIA, mianserine hydrochloride, ISO, isoxsuprine hydrochloride, MER, meropenem, MEC, meclofenamic acid, ETI, etilifrine hydrochloride, HAL, haloperidol, MOX, moxonidine, CHL, chlorphenesin carbamate, PRO, prothionamide, EPI, epitiostanol.</p

Dose optimization and time course of administration of neuroprotective compounds.

<p>A. Dose response of compounds administered at reoxygenation after 2(ISO) and etilifrine hydrochloride (ETI) and 200 µM for chlorphenesin carbamate (CHL). Isoxsuprine was significantly more protective at 1 nM compared to 0.01, 0.1, and 100 nM (*, <i>p</i><0.01). Etilifrine was significantly more protective at 1 nM compared to 0.01 and 0.1 nM (*, <i>p</i><0.05). Chlorphenesin carbamate was significantly more neuroprotective at 200 µM compared to all other doses (*, <i>p</i><0.05). B. Time course of administration of compounds at the optimal dose at 0, 15, 30, and 60 minutes after reoxygenation onset. Isoxsuprine and chlorphenesin carbamate demonstrated no decrease in neuroprotection when administered up to 60 minutes after reoxygenation onset. Neuroprotection by etilifrine significantly decreased when administered at 60 minutes versus time 0 (*, <i>p</i><0.01).</p

Neuroprotection by isoxsuprine hydrochloride in an animal stroke model.

<p>A. Representative TTC-stained brain sections showing differences in infarction between animals receiving vehicle or isoxsuprine hydrochloride. B. Effect of isoxsuprine hydrochloride on infarct volume. Isoxsuprine hydrochloride (1 mg/kg, IV) given at the onset of reperfusion after a 90-minute MCAO significantly reduced infarct volume compared to vehicle (137±18 mm³ versus 279±25 mm³), <i>p</i><0.001. Closed circles, vehicle, closed squares, isoxsuprine hydrochloride, n = 7 animals for each group.</p

Neuroprotective compounds identified through high-throughput screening of the Prestwick Chemical Library.

1<p>Compounds previously investigated in models of cerebral ischemia are italicized.</p

DISC766623_Supplemental_Material – Supplemental material for A High-Throughput Flow Cytometry Screen Identifies Molecules That Inhibit Hantavirus Cell Entry

<p>Supplemental material, DISC766623_Supplemental_Material for A High-Throughput Flow Cytometry Screen Identifies Molecules That Inhibit Hantavirus Cell Entry by Tione Buranda, Catherine Gineste, Yang Wu, Virginie Bondu, Dominique Perez, Kaylin R. Lake, Bruce S. Edwards and Larry A. Sklar in SLAS Discovery</p

Elastomeric Negative Acoustic Contrast Particles for Affinity Capture Assays

This report describes the development of elastomeric capture microparticles (ECμPs) and their use with acoustophoretic separation to perform microparticle assays via flow cytometry.We have developed simple methods to form ECμPs by cross-linking droplets of common commercially available silicone precursors in suspension followed by surface functionalization with biomolecular recognition reagents. The ECμPs are compressible particles that exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, blood serum, or diluted blood. In this study, these particles have been functionalized with antibodies to bind prostate specific antigen and immunoglobulin (IgG). Specific separation of the ECμPs from blood cells is achieved by flowing them through a microfluidic acoustophoretic device that uses an ultrasonic standing wave to align the blood cells, which exhibit positive acoustic contrast, at a node in the acoustic pressure distribution while aligning the negative acoustic contrast ECμPs at the antinodes. Laminar flow of the separated particles to downstream collection ports allows for collection of the separated negative contrast (ECμPs) and positive contrast particles (cells). Separated ECμPs were analyzed via flow cytometry to demonstrate nanomolar detection for prostate specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 μL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis

Savirin inhibits <i>agr</i>-regulated gene transcription and secreted virulence factor production.

<p>(<b>A</b>) Graphic representation by category of microarray results in LAC after 5 hrs of culture with 50 nM AIP1 and 5 µg ml⁻¹ of savirin. Of 205 <i>agr</i>-regulated transcripts, 122 were affected by savirin by 2 fold or greater and p<0.05, n = 3. All major virulence factors are represented; others shown are representative of the category. (<b>B</b>) Effect of savirin (5 µg ml⁻¹) on transcription induced by 50 nM AIP1 in LAC for <i>hla</i>, <i>psm alpha</i>, and <i>pvl</i> relative to 16S at 1 hr and for <i>agrA</i> and <i>agrC</i> at 5 hr. (<b>C</b>) Effect of savirin (5 µg ml⁻¹) on overnight alpha-hemolysin production by LAC. (<b>D</b>) Effect of savirin (5 µg ml⁻¹) vs. vehicle on the capacity of culture supernatant from a clinical <i>agr</i> I blood stream isolate to lyse human neutrophils as measured by LDH release after 2 hr. Data are represented as the mean ± SEM, n = 3 independent experiments. ***p<0.001 **p<0.01, *p<0.05 by two-tailed Student's <i>t</i>-test.</p

Effect of savirin treatment on <i>in vitro</i> host-dependent killing of LAC <i>agr</i>+ and Δ<i>agr</i>.

<p>(<b>A</b>) Percent intracellular survival of LAC <i>agr</i>+ (plus AIP1) or Δ<i>agr</i> treated with savirin (5 µg ml⁻¹) vs. vehicle for 5 hr prior to opsonization and phagocytosis by mouse macrophages (MOI 1∶1). Viable intracellular CFU set at 100% after internalization for 1 hr. Mean ± s.e.m., n = 3 independent experiments performed in triplicate. (<b>B</b>) Log CFU remaining of 1.0×10⁸ LAC <i>agr</i>+ (plus AIP1) or Δ<i>agr</i> treated with savirin (5 µg ml⁻¹) vs. vehicle for 5 hr prior to incubation at pH 2.5 for 2 hr. Mean ± SEM, n = 6. ***p<0.001 **p<0.01, *p<0.05 by two-tailed Student's <i>t</i>-test.</p