X On A Researcher Protocol

Neuron Device Protocol
Xona Microfluidics LLC

27574 Commerce Center Drive, Unit 137
Temecula, CA 92590 USA
Phone: (951) 308 0044 Mon - Fri 10am to 5pm (Pacific Time)
Phone: (855) 234 0044 (Toll Free in U.S. & Canada)
Fax: (951) 346 5566
xonamicrofluidics.com
xonamicrofluidics@gmail.com
© Copyright Xona Microfluidics LLC, 2015 All Rights Reserved
Rev. 30-Jun-2015 Xona Microfluidics, LLC

Table of Contents
A) Getting Started page 1
B) Preparing The Cover Glass page 4
C) Preparing The Devices For Bonding page 5
D) Plasma Bonding The Neuron Devices To Cover Glass page 5
E) Coating Within The Devices With Poly(L)Lysine (PLL)

(For plasma bonded devices only) page 7
F) Coating Cover Glass With PLL

(For non-plasma bonded devices only) page 9
G) Preparing Neurons For Loading Into The Neuron Device page 10
H) Counting The Cells page 11
I) Seeding Neurons Into The Neuron Device page 12
J) Fluidic Isolation page 17
K) Axotomy page 17
L) Staining Neurons In The Neuron Device page 18
M) Supplemental Information page 19
N) Non-Plasma Bonding Supplemental page 20
O) Legal Statements page 23
Rev. 30-Jun-2015 Table of Contents Xona Microfluidics, LLC

A) GETTING STARTED
Thank you for choosing Xona Microfluidics’ patented neuron device. It is our desire that
you be successful in your research and we will do our best to assist you where possible.
Before you begin it is important that you familiarize yourself with the protocols for using
the neuron device.
We also recommend viewing two videos related to using the neuron device published
at jove.com. The videos are also available for viewing on our website. The titles of the
videos are as follows:
Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

(http://www.jove.com/video/305/preparing-e18-cortical-rat-neurons-for-
compartmentalization)
Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

(http://www.jove.com/video/410/non-plasma-bonding-pdms-for-inexpensive-
fabrication-microfluidic)
Proper Storage
The neuron device should be stored in a cool dry place away from volatile chemicals
and direct exposure to intense light. The neuron device is manufactured out of
Polydimethylsiloxane (PDMS), a silicon polymer which can readily absorb contaminants
from its surrounding environment. Properly stored, the neuron device should remain
viable for up to 6 months.
Equipment and Supplies Needed

Corning No. 1 (24 mm X 40 mm) cover glass or suitable equivalent; 60 mm culture
dishes; water bath sonicator; 37ºC 5% CO2 incubator (for cell culture); bio-safety
cabinet; Poly(L)Lysine (PLL) [or Poly(D)Lysine (PDL)]; media and reagents related to your
cells of interest; 3M™ 471 Vinyl Tape, used for cleaning the device (see
http://www.mmm.com to find a vendor near you).
Optional Equipment
Plasma Cleaner/Sterilizer with vacuum pump (available from Harrick Plasma at
http://www.harrickplasma.com/). SPI also sells a plasma cleaner
(http://www.2spi.com/). Other optional equipment: stainless steel cover glass staining
rack (available from Electron Microscopy Sciences at http://www.emsdiasum.com);
cover glass staining dish (also available from Electron Microscopy Sciences); 0.5 mil
thickness Teflon FEP film to help prevent evaporation (available from American Durafilm
at www.americandurafilm.com).
NOTE: Each section contains “Tech Notes”, which provide the researcher with more
specific details and tips about particular procedures.
First Consideration
Rev. 30-Jun-2015 Page 1 of 23 Xona Microfluidics, LLC

You must determine whether you would like to perform plasma bonding or non-plasma
bonding for mounting the neuron device to cover glass. You will have to determine if
you have access to a plasma cleaner or if you intend to purchase one. If the answer is
yes, see the plasma bonding section of the protocol (in addition to glass and cell
preparation).
If the answer is no, you will be performing non-plasma bonding of the neuron device to
PLL or PDL (0.5 mg/ml) coated cover glass. Each method has its advantages and
disadvantages.
Plasma Bonding vs. Non-Plasma Bonding
Plasma bonding advantages: this method creates a permanent irreversible bond

between the neuron device and the cover glass. The surfaces and interior of the
device also become hydrophilic upon exposure to plasma for the first 10 minutes after
plasma bonding, which facilitates the addition of liquids. The seal between the device
and the cover glass is typically tight, thus reducing the chance of leakage. Plasma
bonding also serves to sterilize the device and glass, in addition to cleaning the device
and glass.
Plasma bonding disadvantages: plasma cleaners may be too expensive for some labs
to purchase for the sole purpose of bonding the neuron device to cover glass. The
bond between the device and glass is irreversible, making it impossible to remove the
device for subsequent procedures, such as immunostaining. Please note, however,
that it is normally not necessary to remove the device as immunostaining can be
performed with the device still attached.
Non-plasma bonding advantages: does not require a plasma cleaner, thus saving
new-equipment cost. Devices can be removed from the cover glass after culture, if
necessary. In rare cases it is desirable to remove the device for immunostaining and for
certain microscopy procedures. However, please note that it is possible to stain cells
within the neuron device without removing it from the glass. Also, keep in mind there
will be some cell damage upon removing the device. See our section on immuno-
staining (page 18) for more information on how to minimize this damage.
Non-plasma bonding disadvantages: the cover glass and neuron device remain
hydrophobic, thus the addition of liquids to the device may require the assistance of a
micropipettor to push liquid into the device. If the glass and device are not sufficiently
clean and free of debris, a proper bond may not form, thus leaking may occur.
However, please note, that often in the case of minor leaking around the perimeter of
the wells, the device is still usable. Also, if a proper bond is not formed between the
glass and device, cells and axons may grow under the device rather than through the
microgrooves, in some cases. Additionally, an improper bond can occur if too many
substrates are applied to the glass before the device is attached. Lastly, if the device is
bumped or nudged it could come loose (become unbound) from the cover glass.

Please note, some sections of the protocol pertain only to plasma bonded devices and
some sections pertain to non-plasma bonded devices, while other sections pertain to
both.
Tech Note: Before you start, please review Section I) Seeding Neurons Into The Neuron
Device, (page 12), particularly “Regarding Media and Evaporation” (page 15), for tips
and hints.
Types of Neurons Successfully Cultured In Xona’s Neuron Devices
Cell Type Source Media Substrate

Cortical E18 Rat NBM+B27+Glutamax PLL (0.5 mg/ml)
Cortical E17 Rat NBM + B27 + 500nM PDL (0.5 mg/ml)
L-Glutamine + 1%
antibiotics
Cortical P1-P2 Rat Neurobasal Medium + PLL (1 mg/ml)
B27 + Glutamine +
Pen/strep
Cortical Mouse E14/15 C57/Bl6 Grown in NBM/B27 with 0.001% Poly-L Lysine*
10% FCS for the first 24
hours
Cortical Rat E18 Hooded Wistar Grown in NBM/B27 with 0.001% Poly-L Lysine*
or Sprague Dawley 10% FCS for the first 24
hours
Mixed Glia/Purified Rat/ P1/2 corex or DMEM + 10% FCS 0.0005% Poly-L-
Astrocytes mouse P2/3 cortex Lysine*
Superior Cervical Rat pups post natal Not Specified 0.01% Poly-ornithine
Ganglion day 4/5
Cortical E18 Mouse NBM, 2% B27, Not Specified
Pen/Strep, L-Glutamine
Dorsal Root Neonatal 2-3 days old NBM + B27, Glutamax, PLL (0.1 mg/ml),
Ganglia (DRG) (source not specified) NGF, pen-strep Laminin (0.01 mg/ml)
after device
attachment*
Trigeminal Ganglia Neurons from adult (6- Neurobasal-A 500ug/ml PDLO
8wk) mice medium+B27+2mM (poly-dl-ornithine)
Glutamax+ 7S NGF prior to device
and GDNF attachment and
Mouse Laminin after
device attachment
Cortical, Striatum E18 Rat NBM + B27 and PEI 0.1%
Glutamax
*This customer reports success with this concentration of substrate. However, in order to
ensure proper cell attachment, Xona recommends higher concentrations of substrate due to
medial flow through the device.
B) PREPARING THE COVER GLASS
You may find the following items useful for cleaning and preparing the cover glass:

Glass Staining Dish Stainless Steel
Electron Microscopy Sciences Coverslip Staining Rack
EMS Cat. No. 70312-33 Electron Microscopy Sciences
EMS Cat. No. 72241-01
Tech Note: It is not required to use the staining dish and rack for cleaning the cover
glass. Feel free to adapt a method from your existing lab equipment if possible.
Tech Note: Clean cover glass is essential to your success with using the neuron device,
particularly with the non-plasma bonding method. We recommend Corning No. 1 (24
mm X 40 mm) cover glass, or suitable equivalent.
Tech Note: It is possible to substitute other sizes of No. 1 cover glass, as long as the
substitute is large enough to accommodate the device. It may also be possible to use
standard microscope slides, though it is not recommended. Be sure to consider the
working distance on your microscope and what type of imaging you will be performing,
as this will determine what thickness of cover glass you will be required to use.
Preparation Steps:
1) Place the Corning No. 1 (24 mm X 40 mm) cover glass in the stainless steel staining
rack and place the rack in the glass staining dish. Fill with dH2O.
2) Place the glass staining dish containing the racked cover glass into a water bath
sonicator. Fill the water bath sonicator to the appropriate level and sonicate the glass
slides for 30 minutes.
3) Dispose of the dH2O and rinse the glass slides with 70% Ethanol in the glass staining
dish.
4) Rinse the slides three times with sterile dH2O in the glass staining dish, preferably in a
bio safety cabinet to help maintain sterility.
5) Allow the slides to dry in a bio safety cabinet for several hours, preferably overnight.
6) The slides are now clean, sterile, and ready for plasma bonding. In the case of non-
plasma bonding, the cover glass is now ready for PLL or PDL coating (minimum
concentration 0.5 mg/ml) provided they are contained in a clean environment, such as
a tissue culture hood, and kept sterile.
C) PREPARING THE DEVICES FOR BONDING
The devices supplied by Xona Microfluidics are clean but not sterile. Sometimes in
handling, however, occasional dust and debris will land on the device.

Tech Note: DO NOT use cans of compressed refrigerant air (as commonly used to
clean electronic components) as it has been found to be detrimental to subsequent
cell growth. Any debris remaining on the neuron device can be removed using 3M™
Scotch Brand 471 Vinyl tape.
Tech Note: Once cleaned, it is important to make sure the feature side of the device
remains clean and free from debris. Thereafter, to avoid dust contaminants, the neuron
devices may be housed in a clean, sterile tissue culture dish, with the lid on, and feature
side up until you are ready to use them. The neuron device contains microfluidic
channels and main channels where the cells will be cultured. Be as careful as possible
to not damage the device prior to use.
For non-plasma bonding, we recommend sterilizing the neuron device by rinsing
it with 70% Ethanol and allowing the device to air dry for 30 minutes to 1 hour in a clean
bio safety cabinet. Ensure the device is fully dry before proceeding. This should be done
just prior to cell preparation so that the device is not exposed for more than a few hours
to possible contaminants. Some researchers also prefer to rinse the device with sterile
dH2O after rinsing with 70% Ethanol to ensure all the ethanol is removed. The device
should be allowed to dry feature side up in a biosafety cabinet.
The devices can also be sterilized by autoclaving. The devices can be placed in
a suitable autoclave container and sterilized at a standard setting for packs or wraps.
We have, however, found that some plastic containers can make the devices toxic for
cell culture. Also, an unclean autoclave may impart toxins into the neuron device that
may affect cell growth. Use only suitable autoclavable containers.
Tech Note: If you are plasma bonding the device, the plasma cleaner will sterilize the
device.
Once sterile, the devices are ready for non-plasma bonding to Clean Corning No. 1 (24
mm X 40 mm) cover glass, previously coated with 0.5 mg/ml PLL or PDL.
D) PLASMA BONDING THE NEURON DEVICES TO COVER GLASS
1) Place the neuron device on a suitable tray face feature side up. Place the cover
glass on the tray next to the neuron device.
2) Place the tray containing the neuron device and cover glass 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) Crack the air valve to allow in just a small amount of air.

Tech Note: The pressure should not go above 500 millitorr or below 300 millitorr, as the
plasma will likely not form. Consult your plasma cleaner’s manufacturer instruction
manual for more details.
6) A purple color should appear about 10 seconds after turning on the power and
cracking the air valve – this is the plasma. It may be necessary to adjust the air valve
several times so that the pressure remains between 300 and 500 millitorr. The plasma will
typically start to fade below 300 millitorr and above 500 millitorr. For optimal results,
maintain the pressure between 300 millitorr and 500 millitorr for maximum plasma
formation. This may vary depending on the make, model, and manufacturer of plasma
cleaners, so please consult with the manufacturer for information specific to your
plasma cleaner.
7) Allow the device and cover glass to be exposed to the plasma for 2 minutes (time
and power settings may vary depending on the plasma cleaner). Please note, that
settings and exposure time may have to be optimized depending on your make/model
of plasma cleaner. Some find they obtain a better bond at lower power settings with a
shorter time duration, while others prefer high power for a full 2 minutes.
Tech Note: Plasma cleaning longer than two minutes may cause the neuron device to
plasticize, which may lead to a failure to bond properly.
8) Turn off the power, turn off the vacuum, and open the air valve.
9) Remove the devices and cover glass from the plasma cleaner.
10) In a clean environment (preferably a clean hood or bio safety cabinet) and on a
clean surface previously sterilized with 70% Ethanol, place the neuron device on to the
cover glass. The feature side, exposed to the plasma, should go face down on to the
side of the cover glass also exposed to the plasma.
11) With moderate force, press down on the device evenly to ensure bonding.
12) The device may be housed in a 60 mm sterile tissue culture dish for transport,
incubation, and handing. The device is now ready to be coated with 0.5 mg/ml PLL or
PDL.
Tech Note: After plasma bonding, the device will remain hydrophilic for about 10
minutes. It is important to add liquid, typically 0.5 mg/ml PLL or PDL, to the device within
this time frame.
Tech Note: If more than 10 minutes has elapsed before liquid is added to the device, it
may be necessary to use a micropipettor to push liquid into the device. This can be
done by placing the point of a 200 µl pipet tip at the opening of the main channel and
using it to push liquid into the device.
E) COATING WITHIN THE DEVICES WITH POLY(L)LYSINE (PLL) or POLY(D)LYSINE (PDL)

(For plasma bonded devices only)

Tech Note: The device can be housed in a 60 mm sterile tissue culture dish for transport,
incubation, and handing.
Tech Note: If you are non-plasma bonding, you will instead be coating the entire cover
glass with 0.5 mg/ml PLL or PDL (see Section F) and placing the device on top of the
coated glass. This section is only for those who are performing plasma bonding.
Tech Note: 0.5 mg/ml Poly(D)Lysine, or any other suitable coating material, can be
substituted for PLL for your particular cell culture.
Microfluidic Neuron Device diagram
Above is a diagram of the microfluidic neuron device. Notice how the wells on the Left
(Blue, Cell side) are connected by a main channel as are the wells on the Right (Red,
Axonal side). Each compartment is then connected by microgrooves (black horizontal
bars). When we refer to placing PLL, water, or media in a top well on each side of the
device, what we mean is, for example: Place 150 µl of autoclaved dH2O in one Top
Left (Blue) Well, then place 150 µl of autoclaved dH20 into one Top Right (Red) Well.
The water (PLL, Media, or Cells) will flow through the device microgrooves to the other
corresponding well.
Please note, the designation of the left side as Cell Side and the right side as Axonal
Side is arbitrary and by convention only. Cells could, in fact, be loaded on either side,
or both sides, of the device if desired. For explanatory purposes, it assists us to
designate Left (cell) and Right (axonal) sides of the device, with the device feature side
down.
1) Add 0.5 mg/ml PLL (or PDL) to the top left well of the device and allow to flow
through the main channel into the adjoining well. Wait 5 minutes, then fill the bottom
left well with 150 µl of PLL. Next, add 150 µl of PLL to the top right well. Wait 5 minutes,
then fill the bottom right well with 150 µl of PLL.

2) Place the devices containing 0.5 mg/ml PLL in an incubator at 37°C for a minimum
of 4 hours. Overnight is preferable.
3) After incubation with 0.5 mg/ml PLL, and in a bio safety cabinet, use a 6” or 9” glass
pipette attached to a vacuum line to vacuum out the excess PLL in the wells. Be
careful not to suck all the liquid out of the device. Remove the 0.5 mg/ml PLL from the
wells only. Always leave liquid in the main channels. Be careful to not introduce air
bubbles into the main channels or microgrooves.
4) Add sterile dH2O to the top right well and top left well of the device and allow the
dH2O to flow, via capillary action, into its corresponding bottom wells connected by
the main channel. Once the water has passed through the main channels, fill up the
bottom left and right wells with dH2O.
Tech Note – Device Orientation: Going forward in the protocol and supplementals,
when we say, “place media, cells, water or 0.5 mg/ml PLL in the TOP wells…” we are
referring to the diagram above where the cells will grow on the left (Blue) side and the
axons will grow through the microgrooves into the main channel on the right (Red) side.
Tech Note: On occasion, bubbles may get introduced into the main channels. If this
occurs, use a 200 µl micropipettor with a 200 µl pipet tip to remove the bubbles. Fill the
200 µl pipet tip with 150 µl of sterile dH2O (or media or other suitable liquid) and place
the tip near the opening of the main channel. Next, depress the plunger of the
micropipettor with sufficient force to push the liquid through the main channel. This
may need to be repeated several times in order to successfully force out the bubbles.
5) Aspirate off the water (again being careful not to remove liquid from the main
channels or introduce bubbles) and then add another 150 µl of sterile dH2O to the top
wells of the device (see diagram above) and allow it to flow through to the
corresponding well.
6) Repeat the process two (2) more times and then fill the device with sterile dH2O.
7) Place the device containing dH2O in an incubator at 37°C for a minimum of 1 hour,
then repeat the wash steps again (steps 5-6). Complete this sequence of washing the
device three (3) times with dH2O, with one hour minimum incubation between washes.
Tech Note: Due to the possibility that borate from the 0.5 mg/ml PLL can absorb into
the PDMS, Xona Microfluidics recommends incubating the devices overnight with sterile
dH2O, after several initial quick rinses and (3) 1-hour washes. This will ensure all free
borate will leach out of the PDMS prior to the loading of cells. If not done, there may be
a release of borate into the media over time, which could lead to cytotoxicity.
8) After you have completed washing the device, aspirate off the dH2O and add
Neural Basal Media (NBM) (or other appropriate media) containing the necessary
factors (e.g., glutamax, B27) to the top left well (~150 µl approximate volume) and allow
the media to flow through the device. Wait for 5 minutes and then fill the bottom left

well with 150 µl of media. Next, add 150 µl of media to the top right well. Wait 5
minutes and then fill the bottom right well with 150 µl of media.
9) Aspirate off the media from the wells and refill the top left well with fresh media.
Allow the media to flow into the main channel and then fill the bottom well. Wait for 5
minutes to allow the media to push through the microgrooves. Add 150 µl of media to
the top right well. Allow the media to flow into the main channel then fill the bottom
right well. This ensures the water in the main channels has been thoroughly flushed out.
Incubate the device with media at 37°C for a minimum of 3 hours (or overnight) prior to
loading cells. The device is then ready for loading cells.
F) COATING COVER GLASS WITH POLY(L)LYSINE (PLL)

(For non-plasma bonded devices only)
If you do not have access to a plasma cleaner, you will need to perform the non-
plasma bonding method. For this method, rather than coating with 0.5 mg/ml PLL in the
device, the entire cover glass is coated with 0.5 mg/ml PLL prior to bonding.
Tech Note – Regarding coating the cover glass with 0.5 mg/ml PLL: Coating of the glass
slides with 0.5 mg/ml PLL is crucial. Slides that are not properly coated with 0.5 mg/ml
PLL will cause the neurons to clump or not attach well. In coating the cover glass with
0.5 mg/ml PLL, some researchers simply submerge the cover glass in PLL solution for
several hours. Other researchers insist on storing the clean cover glass in the 0.5 mg/ml
PLL solution indefinitely until just before use (with a minimum of 24 hours contact time).
You may wish to try both methods to see which one is optimal for your research.
1) Clean the cover glass as described in Section B) PREPARING THE COVER GLASS
(page 4).
2) Submerge the cover glass in sterile 0.5 mg/ml PLL and allow the cover glass to
incubate in the 0.5 mg/ml PLL for a minimum of 4 hours. Overnight is recommended.
Typically this is performed in a bio safety cabinet to help maintain sterility.
If a staining rack and dish are not available, the cover glass may be placed in a petri
dish and 0.5 mg/ml PLL poured on top. If this method is used, be sure to keep track of
which side of the glass is the 0.5 mg/ml PLL side.
Alternatively, the clean glass may be racked in a stainless steel staining rack, placed in
a glass staining dish, which is then filled with 0.5 mg/ml PLL. The cover glass can be
stored in the 0.5 mg/ml PLL until ready for use. Again, a minimum incubation time of 4
hours is required, though overnight is recommended.
3) In a bio safety cabinet, rinse the glass slides several times with sterile dH2O.
4) Place the cover glass in sterile dH2O and allow it to incubate for 3 hours.

5) Rinse the cover glass with sterile dH2O several more times, then aspirate off the
water and allow the glass slides to air dry for several hours, or overnight, in a bio safety
cabinet.
G) PREPARING NEURONS FOR LOADING INTO THE NEURON DEVICE
Tech Note: Preparation of neurons for loading into the neuron device may vary
depending on what tissue type and cell type you are using. In the following, we use the
example of E18 cortical rat neurons.
Tech Note: To avoid contamination, ensure all material, reagents, and equipment are
properly sterilized. Be sure to work with the tissue in a sterile, debris-free environment.
1) Obtain (1) E18 rat cortex (2 pieces of tissue) stored in Hibernate E, or suitable media,
on ice.
2) Take 3 long glass pipettes and fire them each to successively smaller sizes using an
alcohol lamp.
3) Remove the tissues from Hibernate E with a glass pipette (with sterile bulb attached)
and place them in 2 ml of Neural Basal Media, B27, and Glutamax (NBM+) in a sterile 15
ml tube.
4) Trituration: Using a sterile bulb and glass pipette, the E18 rat cortex is passed up and
down about 5 to 10 times. Then, the next smaller pipette is used to pipette up and
down the tissue. Finally, the smallest pipette is used to pipette up and down the tissue.
By the end of the process, ensure there are no visible pieces of tissue. (Please see Jove
video, “Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic
Device”, http://www.jove.com/video/305/preparing-e18-cortical-rat-neurons-for-
compartmentalization)
5) Centrifuge the cells down at 1100 rpm for 1 minute.
6) Carefully aspirate the media off the cell pellet.
7) Add 1 ml of NBM+ to the pellet, which is then re-suspended by pipetting up and

down.
8) Pass the re-suspended cells by gravity flow through a cell strainer to remove any
clumps (BD Falcon cell strainer, 40 µm nylon).
9) Count and plate the cells.
Tech Note: During the trituration steps (#4) and pipetting steps (#7), try to minimize
bubble formation, as cell contact with the air can lead to oxidation.

Tech Note: If you are able to perform your own dissection and obtain fresh cortex,
better cell viability may be obtained by omitting Hibernate E and instead placing the
cortex in 0.125% Trypsin in Ca++ Free Mg++ Free dissection buffer.
First, dilute 1 ml 0.25% Trypsin (Invitrogen) with 1 ml of Ca++ free Mg++ free dissection
buffer (final trypsin = 0.125%) into a 15 ml tube. The trypsin buffer is kept ice cold. Place
the tissue into the buffer and incubate immediately at 37°C for 8 minutes. After
incubation, add 10 ml DMEM/10% FBS immediately to the tissue to stop the trypsin
reaction. Centrifuge down the tube, 1 to 2 minutes at 1100 rpm. Remove the
DMEM/10% FBS and re-suspend the tissue pellet in NMB+.
Next, proceed with the cell preparation steps described in G).
Several researchers have found that performing the dissection, followed by immediate
dissociation of the cells, and then loading the devices with the freshly dissociated
neurons, has improved cell viability.
However, if it is not possible to dissociate the neurons immediately upon dissection, then
it is necessary to store the cortex in Hibernate E + B27 at 4ºC. We have found that
neuronal tissue may remain viable up to 48 hours after being stored in Hibernate E + B27
at 4ºC. However, the sooner the neurons are prepared, typically, the greater the
viability is achieved.
H) COUNTING THE CELLS
1) Into a microfuge tube, add 60 µl NBM+, 20 µl of cell suspension, and 20 µl of trypan

blue; then mix.
2) Add about 10 µl of this solution to a hemocytometer and count the live cells.
3) This generally achieves a concentration of about 2.5 million to 4.5 million cells per ml.
This is dependent on the volume in which the cells were re-suspended. Normally the
cells are re-suspended with 1 ml NBM+. This will yield between 2.5 million to 4.5 million
cells per ml. If the tissue was prepared with trypsin, the yield will likely be increased.

I) SEEDING NEURONS INTO THE NEURON DEVICE
For Plasma Bonded Devices
Tech Note: The device can be housed in a 60 mm sterile tissue culture dish for transport,
incubation, and handing.
corresponding well.
down.
1) Aspirate off the media in the wells of the neuron device (from Section E, Step 9).
Ensure media remains in the main channels at all times. Never remove media from the
main channels.
2) Load 20 µl of primary neurons at a concentration between 2.5 to 4.5 million cells per
ml in one well (50,000 to 90,000 cells) on the left side of the neuron device. Check
under a microscope to ensure the neurons are flowing into the neuron device main
channel.

3) Place the device containing neurons into a 37ºC 5% CO2 incubator and incubate
for 10 minutes to allow the cells to attach.
4) Add approximately 150 µl of NMB + to each top well, allowing the media to flow
through the device for 30 seconds to 1 minute, then fill the bottom wells with media and
return the device to the incubator.
5) After 24 hours, perform a media change by removing media from the wells. Add
150 µl of media to each top well, allow the fresh media to enter the main channels for
30 seconds to 1 minute, then fill the bottom wells.
6) Media will need to be monitored daily and changed as needed. The frequency of
media changes is highly dependent on your incubator and your individual culture.
Xona Microfluidics recommends minimizing media changes, if possible. Keep in mind,
however, that evaporation causes salt concentrations in the media to increase, which
is detrimental to cell health, as are pH changes. Frequency of media changes will
need to be determined empirically by the researcher.
Typically, media can be changed every 2 to 3 days, as needed. Partial media

changes can also be performed. Minimizing, or even eliminating, media changes can
be made possible by using secondary containment and/or covering the dish
containing the neuron device with Teflon-FEP film (available from American Durafilm,
see page 15 below, “Regarding Media and Evaporation”).
Tech Note: Some researchers have reported better results from splitting the loading of
cells into (2) aliquots of 10 µl, in other words, placing 10 µl in the top well and 10 µl in the
bottom well. Or, if media flow is too great through the device, a smaller volume of cells
can be used, such as 10 µl instead of 20 µl. In addition, a smaller volume of media can
be loaded to the adjoining well to help slow media flow down. For example, you can
load 20 µl of cells to the top well and add 18 µl of media to the bottom well.

For Non-Plasma Bonded Devices
Required Materials:
Clean and sterile neuron device (see Section C, page 5) and 0.5 mg/ml PLL coated
glass (see Section F, page 9).
corresponding well.
down.
1) The device should be assembled in a clean bio safety cabinet. For efficient
bonding, it is recommended that the glass be placed on a flat surface such as the
stainless steel top in a bio safety cabinet. Just be sure to clean the bio safety cabinet
surface with 70% Ethanol prior to assembling the device. Please refer to our non-plasma
bonding supplemental for more information (see Section N, page 20).
2) Place the device evenly on top of the PLL coated glass and press down firmly but
carefully as not to break the fragile glass. It may be useful to use the back of a pair of

tweezers (previously cleaned with 70% Ethanol) to press down on the device. Pay
attention to key points of the device, such as the areas around the main channels,
microgrooves, and wells, as this is where proper bonding is most needed.
3) Carefully, with sterile tweezers if necessary, lift the cover glass off the bio safety
cabinet stainless steel top and place the cover glass, with the neuron device on top,
into a sterile 60 mm dish. Do NOT attempt to handle the neuron device assembly, since
it is not plasma bonded and may detach from the glass.
4) Add 150 μl of media to the top left well, allow the media to enter the main channel
for 30 seconds to 1 minute, then fill the bottom left well with 150 μl of media. Return to
the incubator for 1 hour. Add 150 µl to the top right well, allow the media to enter the
main channel for 30 seconds to one minute, then fill the bottom right well with 150 µl of
media. Return to the incubator for 1 hour.
5) Remove media from the wells. Seed 20 µl of neurons (50,000 to 90,000 cells, see
Section H, page 11) into the Top Left Well of the device. Immediately inspect the cells
under a microscope to ensure they are flowing into the main channel. Due to the
hydrophobicity of the device and glass, it may be necessary to use the 20 µl
micropipettor to push the cells into the main channel. However, remember that the
device is non-plasma bonded to the glass and it can come off the glass. Thus, be sure
not to bump the device or use too much force. Always handle the device by the glass
slide. Do not pick up the assembly by the device itself or you may break the bond.
6) Place the neuron device contained in a 60 mm dish into a 37ºC 5% CO2 incubator
for 10 minutes so the neurons can attach.
7) After 10 minutes, add 150 μl of media to each top well. Allow the media to enter the
main channels for 30 seconds. Add 150 μl to each of the bottom wells. Return the
device to the incubator. It may be necessary to induce media flow through the
device. This can be done in a similar manner to removing bubbles, however, more
care must be taken since the neuron device is not plasma bonded to the cover glass
and can become easily detached. Place the tip of a 200 µl micropipettor at the
opening of the main channel and dispense media into the device.
Regarding Media and Evaporation
Some researchers swear by not changing the media on their neurons once they are
plated. It is thought that neurons may secrete factors that help keep them healthy and
that by changing the media too often these factors are removed.
Other labs change the media on the cells every 2 or 3 days, or as needed, due to
evaporation. How often you need to change the media in the device will be
dependent on your incubator. Some incubators maintain humidity much more
efficiently than others.
Keep an eye on the media in your devices. Pay particular attention to the level of
media in the wells. If you see this level decrease noticeably, you will need to change

the media. Evaporation of media can increase the salt concentration and lead to pH
variation, which can lead to unhealthy neuronal cultures.
There is a balance between changing the media too often and not changing the
media enough. The best case scenario would be to not change the media at all for
the duration of the culture. Unfortunately, this is not always possible.
One possible way to overcome media evaporation is to wrap the 60 mm dish

containing the neuron device with 0.5 mm thick Teflon FEP film (American Durafilm,
Cat.# 50a). This film minimizes evaporation but allows gas exchange. It is possible to
culture neurons in the device in a 60 mm dish wrapped with Teflon FEP Film for over two
weeks without a media change.
6) Use hydrostatic pressure to ensure media enters into the microgrooves. This can be
achieved by ensuring the volumes in the wells on the left side of the device are higher
than the right side of the device. The difference in volume will create hydrostatic
pressure and force media through the microgrooves. For example, load 150 µl of media
in each left side well and 100 µl of media in each right side well.
Tech Note: Some researchers prefer to load cells immediately after non-plasma
bonding and forgo pre-loading the device with media. Researchers may need to
determine which method works best for them.
Bubbles In The Microgrooves Or Partial Filling
On some occasions researchers may notice bubbles in the microgrooves of the device,
or microgrooves not completely filling, particularly when performing the non-plasma
bonding method. If you observe bubbles in the microgrooves or microgrooves not
completely filling, you can use differential volumes to help alleviate this. Fill the wells on
the cell side full with media (150 µl each well), while placing a smaller volume (~50 µl
per well) on the axonal side. This will create hydrostatic pressure pushing through the
grooves. Place the device in the incubator for 30 minutes and then observe under a
microscope. If bubbles or unfilled microgrooves are still observed, incubate longer, up
to overnight, if necessary.
Tech Note: As with other wash steps, be careful to NOT remove all the liquid. Only
remove liquid from the wells of the device. Always leave liquid in the main channels.
Removing liquid from the main channels will introduce air bubbles and likely lead to
device failure.

J) FLUIDIC ISOLATION
The neuron device consists of two chambers, 100 µm in height, connected by a

microgroove barrier that is 3 µm in height. The hydrostatic pressure between the two
chambers separated by the microgroove allows the researcher to fluidically isolate
each chamber. This is accomplished by keeping the volumes in the wells on one side of
the device higher than the other side of the device. The difference in volume creates
hydrostatic pressure, thus fluidically isolating each compartment.
(Taylor, et al, Nat Methods. 2005 Aug:2(8):599-605)
If properly maintained, fluidic isolation between the somal and axonal compartments
can be achieved for 12 to 24 hours, allowing the researcher to differentially treat either
the somal side or axonal side. Results may vary depending on concentrations and time
frames. See the above-referenced publication for more details.
K) AXOTOMY
The neuron device also makes it possible to perform axotomy, cutting axons via
vacuum aspiration in the distal chamber, and observing their regrowth.
(Park, et al, Nature Protocols. 2128 Vol. 1 No. 4 2006)

Tech Note: To ensure full cutting of the axons, it may be necessary to repeat the
aspiration several times. After the first aspiration, force media into the axonal side’s
main channel and view under a microscope. If axons are still visible, continue to
perform the aspiration axotomy until sufficiently cut. In some cases, axons may be
forced down parallel to the microgrooves. In this case, it may be effective to alternate
aspiration from the top, fill with media, then aspirate from the bottom.
L) STAINING NEURONS IN THE NEURON DEVICE
All procedures can be carried out in the neuron device. Differential volume on each
side of the device is recommended to help move antibody and stain through the
microgrooves.
Day 1:
- Fix cells in fresh 4% paraformaldehyde for 30 min @ RT
- Wash with PBS for 5 min x 2
- Permeabilize in PBS-A (PBS + 0.2% triton) for 30 min @ RT
- Block in PBS-B (PBS + BSA 3%) + 5% goat or horse serum for 30 min @ RT
- 1st antibody in PBS-B (+ 5% serum) overnight, @ 4ºC
Tech Note – Removing the Device Prior to Fixation: Only non-plasma bonded devices
can be removed from the cover glass. Please note, that in many cases it is not
necessary to remove the device, particularly when imaging the soma side and axon
side main channels.
Removing the device becomes beneficial when desiring to image neurites within the
microgrooves. In some cases, the microgroove region does not stain well. Staining with
the device removed can improve imaging of the microgroove region.
However, removing the device can also damage some of the neurites. Neurons and
neurites may also, in part, adhere to the PDMS when removing the device.
To help minimize damage, first remove most if the media in the wells. Lift up two corners
of the device with both index fingers (sort of peeling them up) VERY slowly. With your
middle fingers make sure that the glass is held down. At one point the fluid from the
main channels will seep under the rest of the device. At this point, remove the device
straight up and fix right away. This technique should help minimize damage.
Some researchers chill the device on ice for 5 minutes before removing from the glass.
It is not certain if this is a benefit or not. Some fix cells first, then remove the device, but
often this can lead to more damage as parts of the cells and axons fix to the PDMS and
get torn when the device is removed.
Day 2:
- PBS-B 3x10 minute rinse
- Incubate with the secondary (in PBS-B) for 1-2 hours
- PBS wash 3x5 minute
- Mount in Fluoromount G (30-40 µl per hole, top wells only)

M) SUPPLEMENTAL INFORMATION
For E18 cortical rat neurons, B27, PenStrep, and Glutmax are added to Neural Basal
Media (NBM) (Invitrogen) prior to use. Once mixed, the NBM+ may be stored for up to
a week at 4°C.
NBM For 25 ml Dilution

B27 500 µl 1/50
Pen/Strep 250 µl 1%
Glutamax 62.5 µl 0.25%
Preparation of 0.5 mg/ml Poly(L)Lysine (PLL) solution in borate buffer for coating
coverslips
1.24 g of boric acid and 1.9 g of sodium tetraborate is added to 300 ml of nanopure
water (final volume will be 400 ml) and stirred to dissolve for about 30 minutes. Next, the
pH is adjusted to pH 8.5. Two hundred (200) mg of PLL is added and stirred until
dissolved. The final volume is adjusted to 400 ml. In a bio safety cabinet, the 0.5 mg/ml
PLL solution is then sterile filtered and aliquoted into sterile 50 ml tubes. Aliquots are
stored at -20ºC until use.
Tech Note: Some researchers prefer PLL at a concentration of 1 mg/ml. We

recommend you try both and see what works best for your needs.
Tech Note: Poly(D)Lysine may be substituted for PLL. This is entirely up to the researcher.
Results may vary depending on the cell type.

N) NON-PLASMA BONDING SUPPLEMENTAL
The image below illustrates the recommended way for bonding our devices to cover
glass. The device is held level just above the clean, sterile, and coated (PLL or PDL 0.5
mg/ml) cover glass and dropped onto it. Then the device is lightly pressed on to the
glass.
**Handle the device-glass assembly by the glass only
When handling the device-glass assembly it is important to pick up the assembly by the
glass only. If the assembly is lifted up by the PDMS part, it is possible the bond will be
partially broken. Also, when pressing the device onto the glass, it is important to not put
too much pressure particularly on one side or the other. Too much pressure can
actually break the bond.

Handling by the glass, the glass-device assembly can be turned upside down and
inspected for proper bonding. Areas not bound will appear clear. Bonded areas will
appear grayish in color. The main channels will not be bound to glass and will appear
clear. All the other areas should be bound. If clear areas are noticed near the wells or
near the main channels this could indicate improper bonding.
If cells and media have not been loaded yet then the device can be removed and re-
bonded.
For first time users it may be beneficial to also view the glass-device assembly under a
microscope to ensure proper bonding of the microgrooves. The following images
illustrate improper vs. proper bonding:
Properly bound microgrooves This image demonstrates proper

bonding. Green food coloring
was used for illustration purposes.

A view of a properly bound device
illustrated with green food coloring

O) LEGAL DISCLAIMERS
Xona Microfluidics, LLC makes no guarantees about the researcher’s ability to culture
neurons with the neuron device. It is up to the researcher to establish or use previously
established methods for culturing neurons. Xona Microfluidics, LLC is not responsible for
device leakage or failure due expressly to user error or failure to adhere to the
recommended protocols.
While Xona Microfluidics, LLC can advise that E18 Cortical Rat Neurons after two weeks
of culture will likely have minimal, if any, dendrites crossing over the 450 µm microfluidic
barrier, Xona Microfluidics, LLC cannot guarantee dendrites will not cross the barrier.
Researchers wishing to ensure no dendritic crossing of the microfluidic barrier after two
weeks of culture are recommended to use the 900 µm neuron device.
While Xona Microfluidics, LLC can advise that with E18 Cortical Rat Neurons, axons will
typically begin to cross the 450 µm barrier after 3 to 4 days in culture, Xona Microfluidics,
LLC can make no guarantees that your particular neuron culture will successfully grow
axons through the microgroove barrier at any time point. Axon and neurite length will
depend specifically on the neuron type and viability of the culture, in addition to any
factors added to the neuronal culture that are either stimulatory or inhibitory.
Xona Microfluidics, LLC can advise that, if carried out properly, fluidic isolation can be
achieved between the somal compartment and axonal compartment provided the
volumes between the two chambers are differentially maintained, in addition to all the
other procedures being carried out properly for a period of 12 to 24 hours.
While Xona Microfluidics, LLC acknowledges axotomy can be performed within the
device via vacuum aspiration, Xona Microfluidics, LLC makes no guarantees about
axotomy efficiency.
© Copyright Xona Microfluidics LLC, 2015 All Rights Reserved

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Neuron Device Protocol

Xona Microfluidics LLC

© Copyright Xona Microfluidics LLC, 2015 All Rights Reserved

Rev. 30-Jun-2015 Xona Microfluidics, LLC

A) Getting Started page 1

B) Preparing The Cover Glass page 4

C) Preparing The Devices For Bonding page 5

D) Plasma Bonding The Neuron Devices To Cover Glass page 5

E) Coating Within The Devices With Poly(L)Lysine (PLL)

F) Coating Cover Glass With PLL

G) Preparing Neurons For Loading Into The Neuron Device page 10

H) Counting The Cells page 11

I) Seeding Neurons Into The Neuron Device page 12

J) Fluidic Isolation page 17

L) Staining Neurons In The Neuron Device page 18

M) Supplemental Information page 19

N) Non-Plasma Bonding Supplemental page 20

O) Legal Statements page 23

Rev. 30-Jun-2015 Table of Contents Xona Microfluidics, LLC

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

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

Equipment and Supplies Needed

Rev. 30-Jun-2015 Page 1 of 23 Xona Microfluidics, LLC

Plasma Bonding vs. Non-Plasma Bonding

Plasma bonding advantages: this method creates a permanent irreversible bond

Rev. 30-Jun-2015 Page 2 of 23 Xona Microfluidics, LLC

Types of Neurons Successfully Cultured In Xona’s Neuron Devices

Cell Type Source Media Substrate

B) PREPARING THE COVER GLASS

Rev. 30-Jun-2015 Page 3 of 23 Xona Microfluidics, LLC

C) PREPARING THE DEVICES FOR BONDING

Rev. 30-Jun-2015 Page 4 of 23 Xona Microfluidics, LLC

D) PLASMA BONDING THE NEURON DEVICES TO COVER GLASS

4) Turn on the power to the Plasma Cleaner.

5) Crack the air valve to allow in just a small amount of air.

Rev. 30-Jun-2015 Page 5 of 23 Xona Microfluidics, LLC

E) COATING WITHIN THE DEVICES WITH POLY(L)LYSINE (PLL) or POLY(D)LYSINE (PDL)

Rev. 30-Jun-2015 Page 6 of 23 Xona Microfluidics, LLC

Microfluidic Neuron Device diagram

Rev. 30-Jun-2015 Page 7 of 23 Xona Microfluidics, LLC

Rev. 30-Jun-2015 Page 8 of 23 Xona Microfluidics, LLC

F) COATING COVER GLASS WITH POLY(L)LYSINE (PLL)

Rev. 30-Jun-2015 Page 9 of 23 Xona Microfluidics, LLC

G) PREPARING NEURONS FOR LOADING INTO THE NEURON DEVICE

5) Centrifuge the cells down at 1100 rpm for 1 minute.

6) Carefully aspirate the media off the cell pellet.

7) Add 1 ml of NBM+ to the pellet, which is then re-suspended by pipetting up and

9) Count and plate the cells.

Rev. 30-Jun-2015 Page 10 of 23 Xona Microfluidics, LLC

Next, proceed with the cell preparation steps described in G).

H) COUNTING THE CELLS

1) Into a microfuge tube, add 60 µl NBM+, 20 µl of cell suspension, and 20 µl of trypan

Rev. 30-Jun-2015 Page 11 of 23 Xona Microfluidics, LLC

For Plasma Bonded Devices

Microfluidic Neuron Device diagram

Rev. 30-Jun-2015 Page 12 of 23 Xona Microfluidics, LLC

Typically, media can be changed every 2 to 3 days, as needed. Partial media

Rev. 30-Jun-2015 Page 13 of 23 Xona Microfluidics, LLC

Microfluidic Neuron Device diagram

Rev. 30-Jun-2015 Page 14 of 23 Xona Microfluidics, LLC

Regarding Media and Evaporation

Rev. 30-Jun-2015 Page 15 of 23 Xona Microfluidics, LLC

One possible way to overcome media evaporation is to wrap the 60 mm dish