As ready, these contaminants were unstable when it comes to particle aggregation; adsorption of avidin allowed the elastomeric contaminants to maintain balance during centrifugal washes (2900 g for five minutes) performed in a washing/blocking buffer
As ready, these contaminants were unstable when it comes to particle aggregation; adsorption of avidin allowed the elastomeric contaminants to maintain balance during centrifugal washes (2900 g for five minutes) performed in a washing/blocking buffer. Acoustic Focusing and Separation Acoustic focusing experiments were performed on Nile Red labeled EC em /em Ps (NR-EC em /em Ps) to examine their acoustic contrast properties and the optimal operating conditions of the acoustic sample preparation chip (e.g., particle concentrations, flow rates, resonance frequency, and applied voltage on the actuating PZT). the force imparted on a particle by the standing wave, which allows for size dependent acoustophoretic fractionation of components such as platelets.25 Of interest here is the observation that particles exhibiting negative contrast, such as lipids, can be separated by collecting them as they are driven to the antinode.21,22,23 The primary acoustic force on particles in an acoustic standing wave field can be calculated from the following equations.22,23,27 using PRISM (Graph Pad) version 5.0b. The dependent variable (Y) is Voxilaprevir the median fluorescence intensity (MFI, y-axis), the independent variable (x) is the ligand analyte concentration (x-axis), Kd is the dissociation constant and Fmax is the maximum MFI. IgG-PE Titration in 10% Plasma 5 105 EC em /em Ps Celastomeric particles functionalized with mouse anti-human PSA monoclonal antibodies (Abcam, Cambridge MA)C were incubated with different concentrations, (0, 21, 42, 84, 168, 336, 672 pM) of goat anti-mouse IgG-phycoerythrin (PE) (Abcam, Cambridge MA) in 200 L of 10% Voxilaprevir volume porcine plasma (diluted in the washing/blocking buffer) for 30 minutes with continuous rocking at room temperature. EC em /em Ps were then analyzed in an Accuri C6 flow cytometer without prior washing. Titration in 0.1% Blood, Acoustic Separation, and Flow Cytometry EC em /em Ps Cagain, elastomeric particles (5 105) functionalized with mouse anti-human PSA monoclonal antibodiesC were incubated with different concentrations (0, 21, 42, 84, 168, 336, 672 pM) of goat anti mouse IgG-(PE) (Abcam, Cambridge MA) in 200 L of 0.1 % volume whole porcine blood (porcine blood was diluted in washing/blocking buffer) for 30 minutes with continuous rocking at room temperature. Samples were then flowed (45 L/min) through the acoustic sample preparation chip with the acoustic field on (2.91 MHz; 10 V peak-to-peak supplied to the PZT) and collected through the outlet silicone tubings. Once collected, ligand-bound EC em /em Ps were analyzed in an Accuri C6 flow cytometer without prior washing. Flow Cytometry Gating in Bioassays Flow cytometry (Accuri C6) data on EC em /em Ps was acquired by gating on forward and side scatter parameters to exclude debris and doublets. The median fluorescence intensity of gated EC em /em Ps was used to formulate the binding curves shown within the manuscript. RESULTS Particle Separation Approach Particles (or cells) with different acoustic contrast Rabbit polyclonal to NSE properties can be focused (i.e., acoustically positioned to nodal or antinodal planes) and then separated using an acoustic sample preparation chip with a downstream trifurcation (Figure 1a) (see SI Figure S1 for an image of an actual acoustic sample preparation Voxilaprevir chip).22 After acoustic focusing, laminar flow carries particles continuously into outlet channels at the trifurcation for collection (Figure 1a). The attached acoustic transducer(PZT) has an appropriate size to allow resonance at the frequency (2.91 MHz) that corresponds to a wavelength that is twice the width (252 m) of the acoustic focusing channel (Figure 1b). Thus a resonant acoustic Voxilaprevir standing wave is established in the fluid-filled cavity of the chip and the field exerts a time-averaged force that focuses positive contrast particles (e.g., blood cells) to the center pressure node and negative contrast particles (e.g., elastomeric particles) to the two pressure antinodes at the sides of the channel (Figure 1b).21,22 Open in a separate window Figure 1 (a) Schematic diagram depicting the separation approach for elastomeric negative acoustic contrast particles (white) from positive acoustic contrast particles (e.g., blood cells) (black) at the trifurcation in a silicon acoustic sample preparation chip. (b) Cross-section, Voxilaprevir at dashed line in (a), of the chip, with the positive contrast particles focused at the pressure node and the negative contrast particles focused at the pressure antinodes under an acoustic standing wave field. Note: Images are not drawn to scale. (c) Bright field image and size histogram of PDMS-based elastomeric particles prepared by bulk emulsification. Particle Synthesis Polydisperse elastomeric particles were synthesized using an oil-in-water bulk emulsion process without the use of detergent. The synthesis method is straightforward and allows polydisperse elastomeric particles to be rapidly synthesized (~1 hour) with a bulk concentration of 1 1.3 108 particles/mL. The diameters of particles produced by this method varied from submicron to approximately 21 m in diameter (Figure 1c). As prepared, these.