Frequently Asked Questions About The Axonal Isolation System (AXIS™)

1. What is AXIS™?

AXIS™ is a microfluidic device that allows for easy culture, regulation, and directional differentiation of neuronal cells via a system of growth chambers and interconnected channels. AXIS™ devices physically isolate developing neurites from each other and their respective neural cell bodies. In addition, they provide researchers an opportunity for targeted exposure of neuronal parts to growth factors, drug compounds, or other reagents of biological interest. This ability to selectively control exposure of neural outgrowth makes AXIS™ a powerful platform for any researcher interested in understanding axons, dendrites, somas, and their roles in synaptic formation, neural cell development, differentiation, regeneration, degeneration, and trauma.

2. What substance is the AXIS™ device made from?

The AXIS™ device is made from a polymer called polydimethylsiloxane (PDMS). PDMS is an inert compound commonly found in many commercially available products such as silly putty, hair conditioner, caulk, adhesives, caulk, cosmetics, acqurium sealants, silicone lubricants, and many more.

3. Will the AXIS™ device interfere with microscopy and live cell imaging?

No. One of the many attributes of the AXIS™ device is that it is optically transparent. This allows researchers to view their cells during testing to ascertain the extent of differentiation they are undergoing.

4. Can immunocytochemistry be performed using the AXIS™ device?

Yes. Samples can be stained and imaged with the AXIS™ device still bound to the glass substrate. If necessary, the AXIS™ device can also be removed for staining and analysis. A protocol for immunocytochemistry is given in the test manual.

5. What is fluidic isolation?

The AXIS™ device is a microfluidic device and was specifically designed for this purpose. The microgrooves that are formed between the glass and the device are extremely small (only about 4µm in height and 10µm in width with varying lengths) and only permit a small amount of liquid to flow through them. On each side of the microgrooves are a couple of interconnected chambers- one consists of wells A and B plus the channel between them and the other is composed of wells C and D and the channel they share. Fluidic isolation occurs when the volume in the two chambers are different. For example, if the chamber side with wells A and B are loaded with twice the volume as the chamber with wells C and D then there will be a slow flow of liquid from A/B to C/D. The larger volume in A/B creates hydrostatic pressure between the chambers thereby fluidically isolating each chamber. The fluid in A/B will slowly flow into chamber C/D but virtually none of the fluid in C/D will make it into chamber A/B. This dynamic allows researchers to investigate the affect a drug or compound has on a particular portion of that cell.

6. What if the AXIS™ device leaks once media is added?

On rare occasions media may leak from non-plasma bonded AXIS™ devices. If this happens there are several options that can be tried. First, use sterile forceps and apply gentle pressure at various points around the top of the device. A better seal may form between the glass and the device thereby stemming the flow. If the leak is small and from an area around one of the wells, media can simply be added daily during the course of the experiment to maintain the volume for healthy cells. If the leak is large then add media to the area in the petri dish surrounding the device to equalize the volumes and stem the flow. If cells have not been added when the leak occurs remove the AXIS™ device, rinse it briefly with sterile water, then let it dry. Then it can be retried by placing it down again on a new glass slide. If it continues to leak then the device has likely been compromised and may not work properly.

7. Can AXIS™ devices be used more than once?

It is not recommended for a variety of reasons. The AXIS™ devices that are provided have been specially prepared and cleaned prior to delivery. Upon arrival they require only a quick sterilization prior to use. However, following use they become coated with protein, cell debris, dust, and other things which will interfere with any subsequent attempts at use. Even after extensive cleaning failure rates are extremely high as the used devices do not bond to glass very well and problems such as leakage, broken channels or microgrooves, and even cell migration and/or axon differentiation under the device become the norm.

8. Can protein lysate be isolated from the AXIS™ device?

Yes. However, keep in mind that typically only about twenty thousand cells are loaded in the device during a single test. Thus, the amount of protein lysate that can be obtained will be limited and the amount from the axonal compartment will be even less. It may be possible to pool lysates from several devices to obtain enough test material though. Alternatively, researchers may want to consider using the neurite outgrowth kit (Cat No NS230) that Millipore also offers since greater amounts of cells are utilized, more material may be recovered.

9. Can I autoclave the AXIS™ device to sterilize it?

The AXIS™ device that is provided is clean but not sterile. The protocol for use details how to sterile it using ethanol and that is the suggested sterilization method. That said, yes the AXIS™ device can be sterilized by autoclaving but great care must be taken to ensure that the device is still functional. The AXIS™ device is made of a polymer called PDMS which is known to readily absorb other chemicals into it. Any chemicals that are absorbed could then leach out during cell culture and be toxic to the cells. Therefore, the autoclave itself must be in pristine condition with no chemicals ever being sterilized in it and the surfaces must be thoroughly cleaned prior to each and every use. Furthermore, any plastic containers or bags that the devices are sterilized in could impart toxins into the devices and will need to be scrutinized. Since most labs utilize autoclaves that are considered common use equipment (i.e. anyone could have put anything they wanted into them) we do not recommend their use.

10. Can RNA be isolated from the AXIS™ device?

Yes. However, as with protein extraction, keep in mind that the low amount of cells in the device will generally lead to low amounts of RNA recovered. Much like protein extraction, multiple devices may be used to pool samples to try to get enough material for testing. Again, the neurite outgrowth kit (Cat No NS230) may be a viable alternative.

11. What is plasma bonding?

Plasma bonding is a method to permanently attach the AXIS™ device to the glass substrate. To accomplish this the AXIS™ device and the glass both need to be exposed to plasma gas to activate their surfaces for bonding. To attempt this type of bonding a specialized piece of equipment called a plasma cleaner is required. There are several advantages and disadvantages to plasma bonding and these are detailed in the protocol describing how to perform plasma bonding.

12. Do I need a plasma cleaner?

No. The AXIS™ device can be bound to the glass substrate without using a plasma cleaner. The plasma cleaner is only required if you wish to permanently seal the device to the glass. Each researcher will have to decide if the additional cost to obtain a plasma cleaner is necessary given the advantages and disadvantages of this method.