A two-inlet (one for the oil, one for the aqueous sample) flow-focusing microfluidic mold was prepared with standard soft lithography techniques using SU-8 photoresist (MicroChem Corp., MA, USA) patterned on a 4-inch silicon wafer. A 10:1 mixture of Sylgard 184 polydimethylsiloxane (PDMS) resin (40 g)/cross-linker (4 g) (Dow Corning, MI, USA) was poured on the mold, degassed under vacuum, and baked for 2 hours at 70°C. After curing, the PDMS was peeled off from the wafer and the inlet and outlet holes of 1.5 mm diameter were punched with a biopsy punch (Integra Miltex, PA, USA). The PDMS layer was bound onto a 1-mm-thick glass slide (Paul Marienfeld GmbH & Co. K.G., Germany) immediately after oxygen plasma treatment. Last, the chip underwent a second baking at 200°C for 5 hours to make the channels hydrophobic (32 ). The microfluidic chip details are presented in fig. S11. The aqueous sample phase and the continuous phase composed of fluorinated oil (Novec-7500, 3M) containing 1% (w/w) fluorosurfactant (RAN Biotechnologies, MA, USA) were mixed on chip using a pressure pump controller (MFCS-EZ, Fluigent, France) and 200-μm-diameter polytetrafluoroethylene (PTFE) tubing (C.I.L., France) to generate 0.5 pl of droplets by hydrodynamic flow focusing.
Mfcs ez
Manufactured by Fluigent
Sourced in France, United States
The MFCS-EZ is a flow control system designed for microfluidic applications. It provides precise and reliable control of fluid flow rates. The device features multiple channels for simultaneous flow control and can be operated through a user-friendly software interface.
Automatically generated - may contain errors
Lab products found in correlation
Novec 7500, by 3M (2 mentions)
Su 8 photoresist, by MicroChem (2 mentions)
Whatman, by Cytiva (2 mentions)
Inverted microscope, by Nikon (2 mentions)
22 gauge polyethylene tubing system, by Instech (2 mentions)
High speed cmos camera, by Photron (2 mentions)
3.5 cm cell culture dish, by Corning (2 mentions)
Axio observer 3, by Zeiss (1 mentions)
Ptfe filter membrane, by Cytiva (1 mentions)
Hbo 103w 2, by Osram (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Kolliphor p188, by Merck Group (1 mentions)
Micro slide, by Ibidi (1 mentions)
Eos rebel t6i, by Canon (1 mentions)
Perfluoro 1 octanol, by Merck Group (1 mentions)
M flow sensor, by Fluigent (1 mentions)
Fusion 100, by Chemyx (1 mentions)
Ptfe membrane, by Cytiva (1 mentions)
2 switch, by Fluigent (1 mentions)
Es 20, by Grant Instruments (1 mentions)
44 protocols using mfcs ez
1
Microfluidic Droplet Generation with Fluorinated Oil
2
Microfluidic Cell Culture System
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The flow through the chip was achieved by eight pneumatic controllers (MFCS-EZ, Fluigent, Le Kremlin-Bicêtre, France), which were connected to eight 2 mL sample vials (Biozym Scientific GmbH, Hessisch Oldendorf, Germany), schematically shown in Figure 3 a. The same tubing was used for connecting reservoirs with the microchip. A polytetrafluoroethylene (PTFE) filter membrane (Whatman, Maidstone, UK) between the tube outlets and the microchip ports was used to prevent clogging of the chip by particles bigger than 5 µm. Reservoirs and chip were placed on a fluorescence microscope (Axio Observer 3, Zeiss GmbH, Jena, Germany), surrounded by an incubator to maintain a cultivation temperature of 37 °C. Connecting components were designed and constructed out of PTFE. To avoid leakage, O-rings were placed between the fluidic chip and the PTFE holder, as shown in Figure 3 b.
3
ATPS Droplet Generation in Microfluidics
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Figure 1 shows the experimental setup for ATPS droplet generation including a microfluidic chip and a high-precision microfluidic pressure control system (MFCS-EZ, Fluigent, Inc., USA). The microfluidic chip consists of a flow-focusing geometry with a height of 50 µm, a dispersed channel width (WD) of 50 µm, and a continuous and main channel width (W) of 100 µm. A precise pressure control system includes a pressure pump and two reservoirs for dispersed (salt or DEX) and continuous (PEG) solutions. ATPS droplet generation was observed and recorded using an inverted microscope (IX73, Olympus Corp., Japan) with a 10× objective lens and a high-speed camera (Fastec IL5S, Fastec Imaging Corp., USA). The camera operated at 500 fps with an exposure time of 1.0 ms to capture a series of images at high-speed. ImageJ software was employed for quantification of droplet shapes, droplet sizes, and number of droplets.![]()
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4
Fabrication of PDMS Microfluidic Devices
5
Particle Separation via Deterministic Lateral Displacement
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Imaging was performed by epifluorescence microscopy (Olympus, BX60) with a built-in 100 W mercury lamp (Osram, HBO 103W/2). A standard FITC (fluorescein isothiocyanate) filter cube (Olympus, U-MSWB2) was used to detect fluorescent beads. Imaging was performed with a monochrome fluorescence CCD camera (Olympus, XM10). Sequences of images were superimposed and analyzed with ImageJ software to visualize the trajectory of particles flowing in the DLD channel. Fluids were actuated by a pressure-based flow controller (Fluigent, MFCS-EZ) with input pressures from 50 mbar to 1 bar according to the DLD design in order to obtain a flow rate of about 100 μL/min in the pillar array. The flow rate was chosen to obtain a Stokes flow and avoid flow stream mixing. In the Stokes regime, the flow rate does not influence the DLD separation of particles.
6
Measuring Surface Tension in Embryos
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As described previously [22 (link),64 (link)], a microforged micropipette coupled to a microfluidic pump (Fluigent, Le Kremlin-Bicêtre, France, MFCS EZ) is used to measure the surface tension of embryos. In brief, micropipettes of radii 8 to 16 μm are used to apply stepwise increasing pressures on the cell surface until reaching a deformation, which has the radius of the micropipette (Rp). At steady state, the surface tension γ of the cell is calculated from the Young–Laplace’s law applied between the cell and the micropipette: γ = Pc / 2 (1/Rp—1/Rc), where Pc is the critical pressure used to deform the cell of radius of curvature Rc.
Eight-cell stage embryos are measured before compaction (all contact angles < 105°), during which surface tension would increase [22 (link)].
Fragmented cells and their control cells are measured 10 to 15 hours after fragmentation. At that point, enucleated fragments are mostly irregular in shape and cannot be measured.
Measurements of individual blastomeres from the same embryo are averaged and plotted as such.
Eight-cell stage embryos are measured before compaction (all contact angles < 105°), during which surface tension would increase [22 (link)].
Fragmented cells and their control cells are measured 10 to 15 hours after fragmentation. At that point, enucleated fragments are mostly irregular in shape and cannot be measured.
Measurements of individual blastomeres from the same embryo are averaged and plotted as such.
7
Microfluidic Device Protocol for Sample Separation
8
Microfluidic Dead-End Filtration Characterization
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The schematic of the experimental setup is shown in Fig. 1(a) . For the microfluidic dead-end filtration, a pressure-driven flow was created in the microchannel by using microfluidic flow control system (MFCS) (Fluigent, MA, USA). The feed was connected to the inlet via a flow unit (Fluigent, MA, USA) and permeate was collected from the outlet of the MMM system (Fig. 1(a) ). The MMM filtration experiments were performed at a constant-pressure difference (ΔP), maintained by the microfluidic pressure controller (MFCS-EZ) (Fluigent, MA, USA). The corresponding volumetric flow rate (Q) was measured directly from the flow-rate-control-module software (Fluigent, MA, USA). All experiments were conducted at creeping flow condition (Re < 1) with maximum fluid velocity vmax ~ 6.84 × 10−4 m/s, considering the channel hydraulic diameter dh ~ 2.86 μm. Three different foulants (polymer, particles and a mixture of polymer and particles) were tested at neutral pH condition. All experiments were performed at room temperature and repeated thrice. The MFCS (Flow unit) was never let to dry & cleaned thrice with ethanol solution before changing any feed sample.
9
Microfluidic Water-in-Oil Droplet Generation
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A two-inlet flow-focusing
device was prepared using standard soft-lithography techniques. In
brief, the microfluidic mold was obtained by coating a 4 in. silicon
wafer with SU-8 photoresist (MicroChem Corp.) reticulated upon UV
exposure. Following careful cleaning of the mold using isopropanol,
a 10:1 mixture of Sylgard 184 polydimethylsiloxane (PDMS) resin (40
g)/curing agent (4 g) (Dow Corning) was poured onto the mold, degassed
under vacuum, and baked for 2 h at 70 °C. The PDMS slab was peeled
off the mold, and inlets and outlets were punched using a 1.5 mm diameter
biopsy puncher (Integra Miltex). The PDMS slab was bound on a 1 mm
thick glass slide (Paul Marienfeld GmbH & Co) immediately following
oxygen plasma activation. The chip underwent baking for 5 h at 200
°C to make the channel hydrophobic. Monodisperse water-in-oil
droplets were generated by mixing the aqueous samples and the continuous
phase (fluorinated oil Novec 7500, 3 M + 1% (w/w) fluorosurf, Emulseo)
on the chip using a pressure pump controller MFCS-EZ (Fluigent) and
200 μm inner diameter polytetrafluoroethylene tubing (C.I.L.).
device was prepared using standard soft-lithography techniques. In
brief, the microfluidic mold was obtained by coating a 4 in. silicon
wafer with SU-8 photoresist (MicroChem Corp.) reticulated upon UV
exposure. Following careful cleaning of the mold using isopropanol,
a 10:1 mixture of Sylgard 184 polydimethylsiloxane (PDMS) resin (40
g)/curing agent (4 g) (Dow Corning) was poured onto the mold, degassed
under vacuum, and baked for 2 h at 70 °C. The PDMS slab was peeled
off the mold, and inlets and outlets were punched using a 1.5 mm diameter
biopsy puncher (Integra Miltex). The PDMS slab was bound on a 1 mm
thick glass slide (Paul Marienfeld GmbH & Co) immediately following
oxygen plasma activation. The chip underwent baking for 5 h at 200
°C to make the channel hydrophobic. Monodisperse water-in-oil
droplets were generated by mixing the aqueous samples and the continuous
phase (fluorinated oil Novec 7500, 3 M + 1% (w/w) fluorosurf, Emulseo)
on the chip using a pressure pump controller MFCS-EZ (Fluigent) and
200 μm inner diameter polytetrafluoroethylene tubing (C.I.L.).
10
Microfluidic Flow Control for Physiological Conditions
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The flow velocity in the microchannels
was established through a PC interface by means of a pressure controller
(Fluigent, MFCS—EZ) with a precision of 0.006% of the pressure
range. The output pressure of the controller was connected to the
headspace of a 3D-printed reservoir inserted into the microfluidic
chip inlet with the desired solutions to flow into the chip.35 An analytical solution of the flow velocity
into semicircular channels has been given by Federspiel and Valenti36 (link) as follows with ΔP being the pressure
drop along the channel, r the channel radius, μ
the fluid viscosity, and L the channel length. Thus,
following this expression, the flow velocity in our channels was adjusted
to that of physiological conditions (in the range of 0–1 mm/s)
through the application of a pressure drop by a pressure controller.
Hence, the pressure drop ranged between 1 and 20 mbar to achieve a
mean flow velocity from 0.1 to 0.5 mm/s depending on the microchannel
dimensions (cf.Supporting Information, Figure S4 ).
was established through a PC interface by means of a pressure controller
(Fluigent, MFCS—EZ) with a precision of 0.006% of the pressure
range. The output pressure of the controller was connected to the
headspace of a 3D-printed reservoir inserted into the microfluidic
chip inlet with the desired solutions to flow into the chip.35 An analytical solution of the flow velocity
into semicircular channels has been given by Federspiel and Valenti36 (link) as follows with ΔP being the pressure
drop along the channel, r the channel radius, μ
the fluid viscosity, and L the channel length. Thus,
following this expression, the flow velocity in our channels was adjusted
to that of physiological conditions (in the range of 0–1 mm/s)
through the application of a pressure drop by a pressure controller.
Hence, the pressure drop ranged between 1 and 20 mbar to achieve a
mean flow velocity from 0.1 to 0.5 mm/s depending on the microchannel
dimensions (cf.
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