Isoton 2

PBCA-MB were synthesized based on anionic-emulsion polymerization as described previously [18 (link)]. Briefly, 3 mL of n-butyl cyanoacrylate (BCA, Special Polymers, Sofia, Bulgaria) was added drop-wise to 300 mL aqueous solution containing 1% of Triton-X100 at pH 2.5. This mixture was emulsified by Ultra-Turrax T-50 basic (IKA Werke, Staufen, Germany) at 10,000 RPM for 1 h at room temperature. The resulting solution was centrifuged at 500 RPM (46 G) for 10 min and washed with 0.02% (v/v %) aqueous solution of Triton-X100 (pH = 7, Sigma-Aldrich, Munich, Germany) until the subnatant was transparent. Size and concentration of MB were measured using a Coulter counter (Multisizer 4e, Beckman, Brea, United States). To perform Coulter counter measurements, a 5 μL solution of MB was mixed with 20 mL of ISOTON® II (Beckman Coulter, Brea, United States), and triplicate readings were taken.

Cell cultures were grown overnight to early log phase at 25°C. A 900 ml sample of each culture was fixed with 100 ml of 37% formaldehyde for 30 min and then washed twice with PBS containing 0.04% sodium azide and 0.02% Tween-20. Cell size was measured using a Coulter Counter Z2 (Channelizer Z2, Beckman Coulter) as previously described (Jorgensen et al., 2002 (link)). In brief, cells were diluted into 10 ml diluent (Isoton II; Beckman Coulter) and sonicated for 3 s before cell sizing. Each plot is the average of three independent biological replicates in which three independent technical replicates were averaged.
To assay the rate of cell proliferation on plates, cells were grown overnight in the indicated medium at 25°C and adjusted to an OD₆₀₀ of 0.5. Tenfold serial dilutions were spotted onto YPD or YPG/E containing DMSO or different concentrations of 3-MOB-PP1, 3-MB-PP1 and 3-BrB-PP1 and incubated at 25°C, 30°C or 37°C for 3 days.

Mice were exposed to ozone in a plexiglass chamber (EMB 104, EMMS®) at 1.5 ppm for 2 h, two times a week for 6 weeks. Ozone was created by an ozonisator (Ozonisator Ozoniser S 500 mg, Sander®) and a level of 1.5 ppm was controlled by a sensor (ATI 2-wire transmitter, Analytical Technology®). Mice were euthanized by progressive CO₂ inhalation, 24 h after last ozone exposure and BAL was collected. After a cardiac perfusion with ISOTON II (acid-free balanced electrolyte solution Beckman Coulter, Krefeld, Germany), lung was collected and sampled for analyses.

Two different concepts of hydrodynamic focusing were implemented into the microflow cytometers for efficient and stable particle positioning in high throughput analyses of cells. All components required for hydrodynamic focusing are integrated into the microfluidic chips. The properties and corresponding layouts of microflow channels are summarized in Table 1. Both the two-stage cascade and the spin focusing require only one inlet for the sheath fluid besides the sample inlet and the outlet connection.
Ultraprecision milling allowed fabrication of mold inserts featuring smooth transition between different channel heights. Thus, the flow channel dimensions can be continuously reduced perpendicularly to flow direction and the joining plane. The values given in Table 1 show the difference between cascaded and spin focusing flow stages. The minimal curvature of channel walls was limited by the diameter of the end mill applied. In this case the curvature of joining points between the sample flow channel and sheath flow channel corresponds to 50 μm diameter for both focusing stages in the cascade design. The successive acceleration of cells in the cascaded focusing approach results in reduced shear forces which might destroy fragile cells during hydrodynamic focusing.
The sheath flow rate was adjusted by a pressurized reservoir and controlled by a flow meter (ASL 1430-24, Sensirion AG, Stäfa, Switzerland). Depending on the specific micro flow cytometer used the air pressure applied ranged from about 400 mbar to 800 mbar corresponding to sheath flow rates between 900 μL/min and 1,300 μL/min. Velocities varied from typically 0.5 m/s up to 3 m/s in the center of the flow channel. Ultrapure water was used as sheath fluid when analyzing fluorescent particles or rhodamine dye as sample stream. Isotonic solution (Isoton II, Beckman Coulter GmbH, Krefeld, Germany) was injected as sheath flow in blood cells measurements. The sample flow was controlled by a syringe infusion pump (model 540060, TSE Systems GmbH, Bad Homburg, Germany) using 1 mL syringes. The sample flow varied from 100 nL/min to about 300 μL/min.
The flow characteristics of the microchips were studied by fluorescence microscopy using aqueous solution of rhodamine 6G dye (concentration 30 μmol/L) excited with high pressure mercury arc lamp. The fluorescence of the hydrodynamically focused sample stream was imaged in both directions perpendicular to the direction of flow. Vertical profiles—Perpendicular to the assembly plane of the structure—were imaged via the on-chip integrated mirrors. Optimal image contrast was adjusted by varying the camera exposure time, typically in the range from 100 ms to 600 ms. Image analysis served to quantify the size of the sample stream as a function of the flow rate ratio for the sample and sheath flow (see [10 (link)] for details).
The sample stream is focused perpendicular to the joining plane by dividing the sheath flow in two symmetrical channels. At the sample injection outlet both flows are merged again. The structures shown in Table 1 use two different approaches to focus the sample stream also in vertical direction. The cascade focusing stages exploits significantly different heights of the flow channels for sample and sheath flow (1:8). Compared to single stage focusing, two stage focusing resulted in a considerably increased stability range with respect to the ratio of sample to sheath flow volume rates. To achieve stable conditions, the height of the channels in the cascade was chosen to be 1,000 μm compared to 125 μm flow channel dimension of the flow channel in the measurement region. On the other hand, in the spin focusing design the height of the fluidic channels is 210 μm (Table 1). The principle of operation is illustrated in Figure 2(a). Compared to other microfluidic structures, where up to four separate sheath flows (back, lifting and side sheath) are necessary [27 (link),29 (link),30 (link)] for full control of focusing and positioning of the sample stream only one inlet ensures efficient hydrodynamic focusing. The sample flow exhibits a spinning motion visualized by injecting a dye sample and observing its fluorescence image (Figure 2(b)).