Funding was provided by the Wisconsin Institute of Discovery.

In this report we demonstrate a fluid delivery method that uses small droplet surface tension to pump a desired volume through a microfluidic channel in order to achieve a number of different fluid phenomena. For example, the user may wish to flow a single fluid as fast as possible, or deliver multiple fluids in rapid succession to create specific fluidic patterns. In order to do this, the user must first have an application built around a microfluidic device. The microflluidic device does not need to be bonded, but should be made from a hydrophilic material. Therfore, the method can be utilized with almost any microfluidic device, with performance largely dictated by the geometrical constraints of the microfluidic channel. To help navigate the geometrical constraints of this method, an introduction to the relevant numerical analysis is presented first.
<ol>
<li>Analytical Methods: According to the Laplace Law and the Washburn Law [1], one can relate the flow rate within a microfluidic channel to its dimensions and the properties of the flowing liquid as seen in equation (1), <img src="/files/ftp_upload/1329/eqn1.png" alt="equation 1" />(1) where &Delta;P is the pressure difference between the inlet and the outlet, &gamma; is the liquid surface tension, R is the inlet drop radius, Q is the flow rate and K is the fluidic resistance as described by equation (2), <img src="/files/ftp_upload/1329/eqn2.png" alt="equation 2" />(2) where &eta; is the liquid viscosity, L0 is the channel length, h is the channel height, w is the channel width, &lambda;=w/h and g (&lambda;) =1.5 if &lambda; &gt;4.45 or <img src="/files/ftp_upload/1329/g_lambd.png" alt="symbol 6" /> if &lambda; &lt; 4.45. Substituting equation (2) into equation (1), always assuming that h&lt;w and solving for Q, one obtains equation (3), (3)<img src="/files/ftp_upload/1329/eqn3.png" alt="equation 3" /> The same analysis can be done for the velocity of the fluid inside a channel by knowing that Q=VA, where V is the fluid average velocity and A is the cross-sectional area or hw. Plugging these into equation (3) you come up with equation (4), (4)<img src="/files/ftp_upload/1329/eqn4.png" alt="equation 4" /> An important mechanical concept that is frequently applied in microfluidic biology is shear stress, which relates to flow rate and velocity by equation (5), (5)<img src="/files/ftp_upload/1329/eqn5.png" alt="Equation 5" /> Knowing the relationship between flow rate, velocity and their physical implications as a function of channel dimensions and fluid properties is crucial in the design of a microfluidic device for a given purpose. Once a device is created, the user must then calibrate the fluid delivery system to achieve the desired flow characteristics within the device.</li>
<li>Steps in Setting up and Calibrating Delivery System:
<ol>
<li>Create microfluidic device via soft-lithography technique using polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning) [2]. There are number JoVE articles that illustrate methods for making PDMS microfluidic devices [5]. For this demonstration, we have chosen a simple straight channel, with dimensions as follows: 2.2mm width, 10mm length and 260um height. The inlet and outlet diameters are 1.8mm and 5.1mm respectively (figure 1). Reversibly attach PDMS device to glass slide by pressing it onto a glass slide (or other suitable substrate) and squeezing out any air bubbles [5]. A reversible attachment allows the device to be re-used multiple times. The method can also be used with permanently bonded devices, but it not required.</li>
<li>Fill device with liquid. The hydrophobic nature of PDMS and the hydrophilic nature of glass help move a drop that is placed at the inlet, or outlet, into the channel. If the drop of liquid does not want to go into the channel by itself or if bubbles move into the channel, the user may put a drop of liquid at the inlet, or outlet, and use a pipette at the opposite end to suck the liquid through the channel. Another method of helping the liquid move into the channel is by separating the PDMS device from the glass slide and gently cleaning the PDMS device and the glass slide with ethanol. This returns to the PDMS and the glass slide their hydrophobic and hydrophilic natures, respectively, which may have been weakened with time and use.</li>
<li>After filling device with liquid, place a small drop on the inlet and a bigger drop on the outlet. Make sure passive pumping is happening by watching the small drop at the inlet collapse and observing fluid flow towards the outlet. Again, make sure there are no bubbles inside the channel.</li>
<li>Using The Lee Company&rsquo;s [3] VHS micro dispensing starting kit, put together one or more valves (valve setup in figure 2) consisting of the Lee VHS M/2 24 Volt Valve, a 0.062 MINSTAC Nozzles with orifice size of 0.0100", the Lee 0.062 Minstac to Soft Tube Adapter, the Lee Spike and Hold Driver (for user control, not shown) and the Lead Wire Assembly (connecting the valve to the Spike and Hold Driver, not shown).</li>
<li>An easy way to hold the valves is by using the Bioscience tools miniature holders (figure 2) [4]. These provide a way to precisely aim and hold the valve at a certain position during experimentation by gluing the valve to one end of the holder and using a magnetic base (not shown) on the other side.</li>
<li>Make a reservoir system to be placed a few feet above the PDMS microfluidic device (in our case we used &frac34; ounce syringes open to the ambient, see figure 2). The reservoir provides a pressure head to drive the nozzles, with the pressure being proportional to the height of the reservoir. Alternatively the nozzle valves can be pressurized by any number of different means (ie, compressed gas). Attach a syringe needle to the syringe. A typical syringe needle will easily attach into 1.14 mm inner diameter tubing. The 1.14 mm tubing will then easily attach into 1.58 mm (1/16&rdquo;) inner diameter tubing which then itself connects to the &ldquo;Soft Tube Adapter&rdquo; of the valve. To prevent leakage of liquid in the 1.14 mm to 1.58 mm tubing connection, one may use PDMS as a sealant. Now that there is a line between the syringe needle and the Lee Co. valve, fill syringe reservoirs with liquid. An extra syringe and a valve may be used to help in the purging process (shown but not labeled in figure 2). Place a magnet to the side of the valve; this is how these valves are purged (they are normally closed solenoid valves), and watch liquid start flowing from the reservoir through the valve and out the 0.0100&rsquo;&rsquo; nozzle.</li>
<li>Calibrate system by choosing a valve open time (open time is the time that the valve allows fluid to pass on a per pulse basis) and frequency (number of pulses per second). Activate one valve for a chosen period (a minute or so, just remember the total run time). Weigh the fluid that was delivered from the valve. Knowing the total run time, frequency and per-pulse open time, calculate the grams per millisecond shot out from the valve. This &ldquo;grams per millisecond&rdquo; value will allow you to choose an open time for any desired volume the user may want to be delivered from the valve.</li>
</ol>
</li>
</ol>
Example: System activated for a minute (60 seconds). The frequency was 15 Hz (15 pulses in one second). The per-pulse open time was 20 milliseconds (ms).
(20ms)(15Hz)(60s)=18000ms.
This means that out of the 60,000 ms in one minute, the valve was actually open for 18,000ms. Let&rsquo;s assume the volume of fluid delivered weighed 5 grams. Then,
5 grams / 18000 ms = 2.78e-4 grams/ms.
In the case of water, with its density being one gram per milliliter (mL),
2.78e-4 grams/ms = 2.78e-4 mL/ms.
After calibration, the volume of a drop is dependent on the open time. For example, with an open time of 20ms, and all the parameters remaining the same as in the previous example,
(2.78e-4 mL/ms)(20 ms) = 5.56e-3 mL = 5.56 &mu;L.
To find the open time y needed to make a drop of x microliter (&mu;L) volume,
(x &mu;L) / [(2.78e-4 mL/ms)(1000 &mu;L/mL)] = y ms
8) Aim one or more nozzles to the inlet of the PDMS device (figure 3). Having calibrated the system, calculate the volume coming out of each valve, based on microfluidic device dimensions. For high speed passive pumping (to obtain maximum flow rate), calculate the inlet drop volume necessary to create an inlet drop which possesses a 90deg contact angle with inlet surface [2]. For packet creation, calculate valve frequency and open times and the valve timings necessary to activate two valves in sequence. As seen in Figure 3, the two nozzles can be pointed at the inlet. This can extended to multiple nozzles, all aimed at the channel inlet.
Representative Results:
When properly calibrated, with valve open times correctly calculated and the nozzles properly aimed at the inlet, the user should be able to see flow passively pumped (figure 4). A burst of liquid should come out of the valve and reach the inlet. As liquid reaches the inlet, there is an instant collapse of the inlet drop into the channel, towards the outlet. Liquid within the channel moves only during the collapse of an inlet drop. Complete fluid movement within the channel stops at the end of the drop collapse, providing for instantaneous fluid stop and well defined fluidic boundaries (in the case that the user is flowing multiple liquids). The duration of drop collapse depends on the inlet port radius and the volume of the inlet drop [1]. In our experimental setup and design, inlet drop collapse occurs in a matter of a few milliseconds.
<img border="0" src="/files/ftp_upload/1329/figure1tmb.jpg" alt="Figure 1" /> Figure 1. PDMS microfluidic device with one inlet, left, and one outlet, right. Please <a href="https://www.jove.com/files/ftp_upload/1329/figure1.jpg">click here</a> to see a larger version of figure 1.
<img border="0" src="/files/ftp_upload/1329/figure2tmb.jpg" alt="Figure 2" /> Figure 2. Reservoir system and valve setup. Please <a href="https://www.jove.com/files/ftp_upload/1329/figure2.jpg">click here</a> to see a larger version of figure 2.
<img border="0" src="/files/ftp_upload/1329/figure3tmb.jpg" alt="Figure 3" /> Figure 3. Two valves, both aimed at a single inlet of a microfluidic device. Please <a href="https://www.jove.com/files/ftp_upload/1329/figure3.jpg">click here</a> to see a larger version of figure 3.
<img border="0" src="/files/ftp_upload/1329/figure4tmb.jpg" alt="Figure 4" /> Figure 4. Time-step sequence (33 millisecond) of inlet drop collapse following fluid ejection from a valve. Please <a href="https://www.jove.com/files/ftp_upload/1329/figure4.jpg">click here</a> to see a larger version of figure 4.

<ol>
	<li>Berthier, E., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+rate+analysis+of+a+tension+driven+passive+micropump.">Flow rate analysis of a tension driven passive micropump.</a> Lab Chip. 7, 1475-1478 (2007).</li><li>Duffy, D. C., McDonald, J. C., Schueller, O. J. A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Prototyping+of+Microfluidic+Systems+in+Poly(dimethylsiloxane).">Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane).</a> Anal. Chem. 70, 4974-4984 (1998).</li><li>Harris, J., Lee, H., Vahidi, B., Tu, C., Cribbs, D., Cotman, C., NL, J. e. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-plasma+Bonding+of+PDMS+for+Inexpensive+Fabrication+of+Microfluidic+Devices.">Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices.</a> J Vis Exp. (9), (2007).</li><li>Walker, G. M., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+passive+pumping+method+for+microfluidic+devices.">A passive pumping method for microfluidic devices.</a> Lab Chip. 2 (3), 131-134 (2002).</li></ol>

<ol>
<li>For high speed passive pumping, if the right combination of frequency and per pulse volume (due to the correct open time) is chosen, user should see what seems to be a static drop or shell at inlet and a very fast flow rate inside channel. If overflow occurs, the open time and/or the frequency are too high.</li>
<li>To detect momentum/surface tension interactions, the user should pump one pulse at a time and observe the intra-channel environment while pulse is occurring (from beginning to end). It is recommended by the authors to use...

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<td>Sylgard 184 Silicone elastometer base</td>
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<td>VHS Microdispensing Starting kit</td>
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<li><a href="http://www.theleeco.com">http://www.theleeco.com</a>.</li>
<li><a href="http://www.biosciencetools.com/Catalog/mHolders.htm">http://www.biosciencetools.com/Catalog/mHolders.htm</a>.</li>
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high speed droplet-based delivery system for passive pumping in microfluidic devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

Watch this Scientific Journal Video about High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices at JoVE.com

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

high-speed-droplet-based-delivery-system-for-passive-pumping

Research

JoVE Journal

Biology

13.7K 次观看.  University of Wisconsin-Madison. 一种新型的微流体系统已经开发使用的被动式抽水的现象和用户控制流体输送系统。这种微流体系统已在多种生物应用由于其成本低，易于使用，体积精度高，速度快，重复性和自动化的潜力。

视频: High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

A Tactile Automated Passive-Finger Stimulator (TAPS)

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering controlled spatially structured stimuli to the skin. Consequently, investigators often apply tactile stimuli manually. Manual stimulus application is time consuming, requires great care and concentration on the part of the investigator, and leaves many stimulus parameters uncontrolled. We describe here a computer-controlled tactile stimulus system, the Tactile Automated Passive-finger Stimulator (TAPS), that applies spatially structured stimuli to the skin, controlling for onset velocity, contact force, and contact duration. TAPS is a versatile, programmable system, capable of efficiently conducting a variety of psychophysical procedures. We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.
Corresponding Author: Daniel Goldreich

A Tactile Automated Passive-Finger Stimulator TAPS

We describe a computer-controlled device for investigating the sense of touch: the Tactile Automated Passive-finger Stimulator (TAPS). We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering ...

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 &mu;m diameter and 150 &mu;m depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 &mu;m in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...