Dynaflow® Resolve Faq
Are there any requirements regarding instrumentation, i.e. does this system work with any conventional patch clamp set-up?
Yes, the Dynaflow® Resolve technology fully integrates with conventional patch clamp set-ups and with most inverted microscopes. Fluicell provides everything needed to run the Dynaflow® Resolve platform.
The Dynaflow® Resolve platform includes:
- The Dynaflow® Resolve chip and a mounting socket,
- A motorized scan stage with stage controller and a joystick,
- A syringe pump,
- A lid with connector and tubing to connect to the syringe pump,
- The Dynaflow®Commander software.
No, this type of chip will not work with an up-right microscope.
No, the base of Dynaflow® Resolve chip is made of 0.7 mm thick glass requiring the use of a long working distance air objective.
No, but the chip is very easy to fill with a manually operated syringe.
About 10 minutes, depending on how fast you perform the patch clamp procedure.
The lid is used directly to connect pressure source to all the channels. The plastic lid can easily be opened and resealed to the chip using two nuts and a rubber ring sealing. When the lid is attached, it encloses a common volume of air over all substance wells. By pressurizing this air through the lid, the flow is driven from each well and out into the recording chamber. Additionally the lid functions as a protective barrier, shielding both the user and the solutions.
3 kPa positive pressure is needed when using standard settings.
More details can be found in the Thesis “On Microfluidics in Biotechnology: New Devices and Applications” (Johan Pihl), p. 18-19.
The substance reservoirs hold 150 µl. This is sufficient for 90 min flow time with a flow-rate of 26 µl/min.
The flow rate is set to be approximatively 4 mm/s at the channel outlets.
The chip is composed of a glass microfluidic component and a plastic interface (PEEK) forming the wells and the recording chamber. The plastic interface has been treated with titanium first, then with silicon oxide to allow for even wetting. The bottom of the chip is made of hard anodised aluminium. The lid is made from polycarbonate, O rings and seals from Viton and tubing in Teflon. We are using material that is standard for biological applications.
Using a glass chip has several advantages such as reusability, reducing risk for non-specific binding of “sticky compounds” and non-absorption of highly hydrophobic compounds.
Yes, The Dynaflow® Resolve chip is reusable meaning that the chip is emptied and washed properly after every use.
The Dynaflow® Resolve chip needs to be washed after every use to remove any traces of components such as cell debris, particles, potential salt crystals, all of which can contaminate or block channels.
Overtime, regular cleanings are not sufficient to remove all traces of components within the chip and an annual refurbishment is necessary to maintain the chip.
This is not a problem when the cell is in the collimated flow zones, where only the substance that flows out of the channel is present. But, the cells present in the recording chamber will be affected by the compounds which flow out. If the flow has been running during a whole scan it is advisable to add new fresh cells. Additionally, the chamber should be rinsed thoroughly before applying new cells.
During which phases of the drug development process is the system applicable? Characterization and validation.
Concentration response curves, (EC50, IC50), receptor kinetics, receptor activation, thermodynamics of ligand binding.
Dynaflow® Resolve is used for target identification and validation, lead identification and optimization and preclinical assays.
Yes, it is possible to screen substances with or without re-sensitisation of receptor responses.
The number of substances one can screen per day with the Dynaflow® Resolve system depends very much on the character of experiments. As the chip is reusable, many assays can be performed per day.
Using the Dynaflow® Resolve technology you have unprecedented control of the following parameters:
– The composition of the solution surrounding the cell,
– The time the cell spends in the exposure zone.
By adding agonist free buffer in every other channel of the chip, an intermittent wash out between each agonist is established. The scan stage is easily programmed so that the time the cell spends in the rinsing buffer would be sufficient for reactivation of the ion channel receptors.
Both voltage and ligand gated ion channels, the system is very flexible.
Experimental control is an important issue. With the Dynaflow® resolve system, you always know the true molecular composition of the bioactive substances around the patch-clamped cell, as well as the exposure time and switching speed.
The cell should fit with in the diffusion zones of the collimated flow, and the size and shape of the cell determines the forces acting on the cell by the flow.
Apart from this, as long as a cell can be patch clamped and lifted, it can be employed together with the Dynaflow® Resolve system.
With the present system, the cells need to be dislodged from the surface if they are adherent. We recommend a few different methods for use such as; treatment with Ca2+ free buffers, trypsin or simple mechanical dissociation.
It is OK as long as the user feels comfortable with it. An internal control substance should always be used.
In our experiments, we have seen an effect of more stable seals and higher resistances when the cell is in the flow, due to the flow field pushing the cell against the tip, indicating that the seal becomes better using Dynaflow® Resolve System. Experiments have been performed for over 20 minutes.
Data suggests that the seal becomes stabilized by the flow which means increased stability in the measurements. However, viscous drag could become apparent at high acceleration of lateral movement.
Is the cell negatively affected, seen from a physiological point of view, by long measurement times?
This does not differ from conventional patch clamp and depends to a large extent on which ion channel systems are studied. Some ion channels are reconstituted in membrane without loss of function. Others need an “intracellular cocktail” in the buffer to function properly.