What is recovery?
The recovery of a particular substance is defined as the concentration in the dialysate expressed as percent of the concentration in the interstitial fluid. Relative recovery will approach 100% as the flow rate approches zero, and decrease as the flow rate increases. It is commonly expressed in percent. Absolute recovery is defined as the mass of a substance recovered during a defined time period. It is zero when the flow rate is zero, and will reach a maximum at higher flow rates.
What factors affect recovery?
The absolute recovery (mol/time unit) of a substance from the tissue depends on (1) the “cut off” of the dialysis membrane (usually defined as the molecular weight in Daltons at which 80% of the molecules are prevented from passing the membrane), (2) the length and diameter of the membrane, (3) the flow rate of the perfusion fluid and (4) the diffusion coefficient of the compound through the extracellular fluid. Other factors such as pH of the medium and degradation of the substance may also affect the recovery. The reverse holds true for substances entering the tissue from the probe.
How do the membrane length and flow rate affect recovery?
A longer membrane and a lower flow rate will give a higher recovery.
How should the perfusion fluid be composed?
Ideally the composition of the perfusion fluid should be as close as possible to the composition of the extracellular fluid. However, you may want to change the concentration of sodium, potassium or calcium in order to influence the membrane function in the region you are studying. CMA offers Perfusion Fluid T1 for peripheral tissue and Perfusion Fluid CNS for brain tissue.
What is the pH of the CNS perfusion fluid?
The CNS perfusion fluid is intentionally not buffered to allow it to equilibrate to the same pH as the brain’s interstitial fluid. The pH of a non-buffered solution varies between 5 and 8.
How does the pH of the CNS perfusion fluid impact the tissue?
Since our perfusion fluid is unbuffered it minimally impacts the pH of the tissue. The perfusate will assume the same pH as the surrounding tissue. However, buffering agents within the interstitial fluid may diffuse across the probe membrane at different rates. This can have a small effect on the pH of the tissue.
What perfusion flow rate should be used?
Use a high flow rate if you want to remove or introduce as many molecules as possible per time unit. Use a low flow rate when you want to obtain a more concentrated dialysate (high recovery). Note that a low flow rate gives smaller volume. Consider also the volume needed for the analysis.
What time is needed to obtain steady state conditions?
The introduction of a probe into the tissue will always cause damage and the recovery of cellular function will not be immediate. An hour is often used to reach “baseline conditions”.
When using a probe membrane with a cut-off of 20,000 Daltons, why isn't the recovery for molecules with 20,000 Da molecular weight 100%?
The ability of molecules to pass the membrane decreases logarithmically with increasing molecular weight. By experience we know that most substances with a molecular weight up to 5,000 Da can be dialyzed when using a 20,000 Da membrane. This is of course very dependent on the substance and the sensitivity of the analytical method.
What causes faulty flow?
You may not have calibrated the pump to the syringe you are using. The syringe might be leaking or you are not using the syringe recommended by CMA.
What causes faulty volume of the microdialysis sample?
The probe may be leaking, the tubing may be blocked, something may be wrong with the pump, or the tubing adapters may be leaking.
Why is my recovery so low? Relative Recovery
The concentration recovered will never be similar to what you see in your media. Unless there is total equilibration between your media and the perfusate, the concentration of a given substance in the dialysate will be lower than its actual concentration in your solution. This applies to in vivo and in situ experiments.
The relationship between dialysate and tissue (or solution) concentration is termed "relative recovery" and is defined as the dialysate/interstitial concentration ratio expressed as a percentage:
Relative recovery= Cmd/Cint=1-exp-K0 A/F
where Cmd and Cint are respectively the concentration of your analyte in the microdialysate and interstitial (tissue or solution), K0 is the average mass transfer, A is the membrane surface and F the dialysate flow rate.
As you can see, increasing the membrane surface or decreasing the flow rate will give you a higher relative recovery as well as membrane pore sizes. However, since full equilibration does not occur across the microdialysis membrane, the concentration of the measured substances in the dialysate is never equal to the true interstitial concentration.
If you need to know the interstitial concentration, it can be calculated using other methods, such as the no-net-flux-method.
Microdialysis Probes and Guides
Which length of microdialysis probe membrane should I use?
A longer membrane gives a better recovery of your target molecules but the choice is usually limited by the size of the biological structure you want to study. CMA offers a variety of probes with different membrane lengths from 1 mm to 10 mm, suitable for most experiments.
How do I choose the best membrane material for my study?
Different membrane materials have different molecular weight cut-offs (MWCO). A membrane with a low molecular weight cut-off purifies your sample by excluding large molecules. A membrane with high cut-off recovers large substances such as peptides or smaller proteins. Some substances can also bind to the membrane material. To optimize your choice you should conduct an in vitro test with the substance you want to monitor, but as a general guideline the cut-off should be at least 3-4 times larger than the molecular weight of the target molecule. CMA offers membranes made of polyarylethersulfone (PAES), 20,000 Daltons MWCO, cuprophane, 6,000 Daltons MWCO and polyethersulfone (PES), 55,000 or 100,000 Daltons MWCO.
How do I choose the right microdialysis probe for my experiment?
There are many considerations; however, a stiff probe is suitable for a stereotaxic experiment on the brain while a flexible probe may be suited for microdialysis in a peripheral organ such as adipose tissue, muscle, liver or kidney. CMA probe types are optimized for various uses:
- · CMA 12—optimized probe for CNS use, ideal for chronic implantation
- · CMA 11—a thin stiff probe for discrete brain regions
- · CMA 7—an extremely small stiff probe for CNS studies in smaller animals such as mice with a 6 kDa cut-ff
- · CMA 8—an extremely small stiff probe for CNS studies in smaller animals such as mice with a 20 kDa cut-off
- · CMA 20—a soft non-metallic probe for peripheral tissues and blood vessels
- · CMA 30—a linear probe suitable for skin and other peripheral tissues with a 6 kDa cut-off
- · CMA 31—a linear probe suitable for skin and other peripheral tissues with 55 kDa cut-off
Please use our Probe Selection Guide or contact Technical Support for more help.
Are CMA probes reusable?
CMA probes are only guaranteed for a single use. It is up to the user if they would like to attempt additional uses.
Probes should be rinsed and stored in deionized water between experiments and with each use of the probe the membrane becomes less and less functional.
When are probe guides needed?
Probe guides are used when performing microdialysis in the brain of a freely-moving animal and provide a way to accurately position the probe at the desired stereotaxic coordinates.
In use, the guide is pre-implanted and secured to the animal's skull with dental cement and anchor screws. The animal is then allowed to recover. On the day of the experiment the "dummy" probe within the guide is removed for insertion of the microdialysis probe.
How do I prepare and clean FEP Tubing?
When using FEP tubing, cut the tubing with a sharp scalpel to ensure the ends remain open to flow. After use, flush and rinse the tubing with deionized water to wash out the salts.
How do I prepare Tubing Adapters for use?
Tubing adapters are used to create air-tight, zero internal volume connections between various components of the microdialysis system. To prepare the tubing adapters are soaked in 70% ethanol. This causes the adapters to swell for insertion of FEP tubing and the inlet/outlet lines of other components in the system, such as swivels, probes, syringe needles, etc. The adapters will then shrink back in air to form a secure seal.
What is the difference between the CMA 4004 pump and the CMA 402 pump?
CMA 4004 has a pulse free flow from 0.54 pl/min to 11.70 ml/min and runs four syringes simultaneously. The pump is calibrated for various sizes of syringes. Apart from microdialysis experiments, it can be used for microinjections of preset volumes that can be repeated in intervals.
CMA 402 has flow rates between 0. 1µl/min and 20 µl/min, and is run by two syringes individually (1, 2.5 or 5 ml). Start/stop and flow rate can be set independently for each syringe.
Both pumps can be used for infusion and withdraw.
What is the difference between the CMA 142 and CMA 470 Fraction Collectors?
The CMA 142 collects 20 fractions from 1 probe or 10 fractions from 2 probes. Fraction volumes from can be 1 µL to 50 µL in open vials.
CMA 470 is a refrigerated fraction collector. It can collect 64 fractions from 1 up to 4 probes simultaneously (16 fractions/probe = 64 total). Fraction volumes can be from 1 µL to 2000 µL. Both opened and sealed vials can be used.
What is the use of a CMA 120 System for Freely Moving Animals?
This system enables microdialysis studies on conscious, small laboratory animals over a long period of time. It can be used in combination with any one of CMA’s instruments.
What are the dimensions of the bowls for the CMA 120 System?
The CMA 120 Bowl with Food and Water Containers (8309672) and CMA 120 Plastic Bowl (8309031) are 360 mm high and 400 mm in diameter.