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Millipore Technical Publications


Protocol: Plasma Bonding: An Alternative Protocol
Catalogue Number:
Year:2009
Rev Date:12/01/2009

Another method that can be utilized during testing with AXIS™ devices is plasma bonding. Exposure to plasma gas causes surface activation of AXIS™ devices which can be utilized to form permanent seals to a glass substrate. Not surprisingly, there are advantages and disadvantages of plasma bonding versus traditional (i.e. non-plasma) bonding that a researcher will have to weigh as they decide which method to perform. Advantages of plasma bonding are essentially threefold. First, formation of an irreversible bond between the AXIS™ device and the glass substrate tends to minimizing the risk of leakage. Second, after treatment a temporary conversion of the device from a hydrophobic state to a hydrophilic state occurs thus enabling the addition of protein coating solution. Finally, the last advantage is that plasma treatment will sterilize both the AXIS™ device and the glass substrate if performed properly. Plasma treatment does have a few potential disadvantages as well though. The first is that it requires specialized equipment (a plasma cleaner) to perform the bonding. Optimization of the plasma treatment may also be required to obtain a strong seal between the glass and the AXIS™ device. Second, the irreversible nature of the bond makes it impossible to remove the device for subsequent procedures such as microscopy. This may or may not be an issue depending upon the test objectives. As noted elsewhere the device does not have to be removed for viewing or immunocytochemistry but the clarity of the pictures are typically better without it. Furthermore, the degree of staining of axons in the microgrooves is sometimes reduced as compared to that in the channels (likely due to decreased fluid flow). Since plasma bonded devices cannot be removed it may be difficult to get equivalent staining intensity on all cellular structures. Finally, confocal imaging with plasma bonding may be more challenging as the AXIS™ devices will have to be sealed to coverslips. Working with coverslips can be a challenge since they are more fragile and may require special stage setups on the microscope to hold them.

Below is the protocol for plasma bonding of AXIS™ devices to glass. Please note that the glass should be cleaned using a sonicator (steps 1 and 2 in the glass preparation section in the standard protocol) prior to use. Some optimization of the protocol may be necessary depending upon the make, model, and type of plasma cleaner utilized. Please consult your manufacturer instructions for more details prior to use.

1.Place the AXIS™ device on a suitable tray with the imprinted side face up. Place the glass substrate on the tray next to the device. If desired, multiple devices can be treated simultaneously.
2.Place the tray into the plasma cleaner.
3.Close the air valve, turn on the vacuum pump, and evacuate the chamber to between 300 and 500 millitorr.
4.Turn on the power to the plasma cleaner.
5.Open the air valve slightly and adjust it to let in a small amount of air. The pressure should not go above 500 millitorr or below 300 millitorr or plasma will not form.
6.After about 10 seconds a purple color should begin to appear. Continue to monitor and adjust the air pressure to keep it between 300 and 500 millitorr.
7.Allow the AXIS™ device and the glass to be exposed to plasma gas for 2 minutes.
8.Turn off the power. Turn off the vacuum. Open the air valve.
9.Remove the tray from the plasma cleaner and immediately place it into a laminar flow hood. Ideally the cleaner should be directly adjacent to a hood to limit the risk of contamination.
10.Using sterile forceps transfer the glass to a sterile petri dish (keep the plasma treated side face up).
11.Then transfer the AXIS™ device onto the glass. Make sure that the imprinted side of the device is placed face down.
12.With gentle force press down on the device with the forceps in several locations to ensure even bonding to the glass. Quickly repeat steps 10 to 12 for all devices exposed to plasma gas.
13.The device is now ready for the addition of coating solution. The AXIS™ device will remain hydrophilic for about 10 minutes after the plasma treatment thus it is important to add the liquid within this timeframe.
14.Add 200µl of coating solution to well A (see standard protocol for different coating solutions and concentrations). The solution should enter the channel between well A and B as well as into the microgrooves fairly easily. If necessary, the liquid can be pipetted up and down to aid in the flow. If more than 10 minutes since plasma treatment has elapsed prior to adding the coating solution then a micropipettor may have to be used to force the liquid into the channels and microgrooves.
15.Add 200µl of coating solution to well B.
16.Incubate for 15 minutes at room temperature. At this point check the device on an inverted microscope to verify that the media has flowed across the microgrooves. If it does not flow to the opposite side of all of the microgrooves then pipet the solution up and down alternating between well A and B several times and/or incubate it longer.
17.Once the coating solution is across the microgrooves then add 100µl of coating solution to well C. The solution should flow through the main channel and into well D rather quickly. If it does not, or if any air bubbles appear in the channel, simply pipet up and down a couple times in well C at the opening to the channel.
18.Once the solution is through the channel between well C and D then add 100µl of coating solution to well D.
19.Incubate for 2 hours in a tissue culture incubator or overnight at 4°C.
20.Vacuum aspirate the coating solution from the wells but do not remove the solution from the channels. This will help minimize air bubble formation.
21.Add 200µl of sterile Milli-Q™ water to well A and to well C. Allow the water to flow through the channels and into the other wells (B and D respectively) for 2-3 minutes.
22.Add 150µl of sterile Milli-Q™ water to wells B and D.
23.Aspirate the water from the wells but do not remove the liquid from the channels.
24.Repeat the water wash process at least 2 more times. Some coating substances can be toxic to cells and may require additional washes to completely remove unbound material.
25.Fill the device with sterile Milli-Q™ water as above then place in the tissue culture incubator for a minimum of 1 hour.
26.Aspirate the water from the wells. Do one additional rinse with sterile Milli-Q™ water then aspirate the water from the wells.
27.Add 200µl of media to well A and to well C. Allow the media to flow through the device for 5 minutes.
28.Add 150µl of media to wells B and D.
29.Aspirate the media from all four wells and from the channel between wells A and B. The wash with media ensures that the water in the channels has been flushed out prior to adding cells.
30.Load 5µl of cell suspension (3 to 5 million cells per ml) into the channel between wells A and B.
31.Place the AXIS™ device into a tissue culture incubator for ~10 minutes to allow cell attachment.
32.At this point it is important to check the cells to verify if an acceptable number of cells are present in the channel between wells A and B. If more cells are desired then add another 5µl of the cell mixture to the same channel between wells A and B and incubate for an additional 10 minutes. The optimal amount of cells to plate is user defined and dependent upon variables such as cell type, viability, media, extent of differentiation, etc.
33.Add 100µl of media to well A and then to well B as quickly as possible. Repeat this step so that both wells A and B have a total of 200µl of media each. Adding media to both wells in rapid succession will limit the amount of liquid flow through the channel minimizing cell displacement.
34.Add 100µl of media to well C and then 100µl to well D.
35.Place the device containing neurons into a tissue culture incubator to promote cell growth and/or differentiation overnight.
36.Monitor the cells and the media as discussed above in the standard (non-plasma bonded) protocol.