Frequently Asked Questions
Medium Back to top
1. Once I mix the B27 supplement / Neurobasal media /
0.5 mM glutamine, how long is the media stable?
We store at 4oC in the dark for up to 1 week.
2. How often do I need to resupplement with
Neurobasal/B27/glutamine?
We change 1/2 the medium every 3 to 4 days, but this partly
depends on cell density. See INVITROGEN (LTI) FOCUS 16:6.
If cell density is higher than 500/mm2 ,change 1/2 every 2
days.
3. Can I dilute the B27/Neurobasal + glutamine + glutamate
that came with the BrainBits with some B27/Neurobasal
+ glutamine (no glutamate), say to a 1:2 dilution and
still have the cells grow?
Yes, but not as well. See the above cited FOCUS article.
4. You typically add 25 uM glutamate to the Neurobasal/B27
up to the 4th day in culture. What is the purpose of the
glutamate?
It helps the hippocampal neurons sprout and promotes viability.
5. Is there a problem if glutamate is left out?
You will get about 30% lower survival at 4 DIV.
6. Do I need to have some media or reagents to maintain the
cultures?
You will need coated glass or plastic substrates of your
choosing. The order includes enough medium to start
cultures and maintain them for 4 or 5 days. Beyond that,
you will need more medium, which is available from Invitrogen
(GIBCO).
7. Should I be supplementing the B27/Neurobasal medium
with antibiotics/antimycotics? eg: pen/strep/fungizone
or gentamicin?
We usually culture without antimicrobial antibiotics and anti-
mycotics with good success. This has three advantages.
First, when a contamination arises, it is easier to identify the
source of the contamination. Second, if you use antibiotics,
when you get an infection, it is harder to get rid of. Third,
antibiotics have been shown to activate epileptiform bursting
activity in neurons.Nevertheless, we sometimes start our
cultures in gentamicin (10 ug/ml) and rinse it away after one
hour, after the cells adhere.
*Hibernate, Neurobasal and B27 is for research purposes only and is manufactured by Invitrogen Corporation. Hibernate, Neurobasal, and B27 is a trademark of Invitrogen Corporation.
1. Can I use Hibernate for papain treatment?
BrainBits LLC supplies 5 ml Hibernate-Ca without B27 for
optimum dissociation of BrainBits with papain in a 15 ml
centrifuge tube, sterile. Once you add papain (Worthington),
dissolve at 37oC for 10 minutes. You will need to sterilize the
solution with a 0.2 um filter.
2. Do you always add B27 glutamine to Hibernate or does it
still keep the cells alive in just Hibernate?
Almost always. Please see Figure 1 of Brewer and Price
(1996) PM:8856709 At 37 degrees C in
ambient CO2, the LD50 in Neurobasal/B27/gln is <6 hr., in
Hibernate/gln without B27 is >24 hr., in Hibernate/B27/gln
is >3 days.
3. Can we supply Hibernate without glucose and without
pyruvate?
Yes, in fact we have several customers who have made this
request. So, we can supply Hibernate A without glucose
and without pyruvate at the same price. You need only to
tell us whether you want the osmolarity adjusted to the
regular level with NaCl or left lower than usual so you can
make your own adjustments.
4. Other uses of Brainbits include:
Substrate Back to top
1. I don't use poly-D-lysine very often. I made a (100x) stock
solution of 5 mg/ml and aliquoted it into small cryovials.
How long can I store the stock solution frozen and still
have it work with the hippocampal cells?
Probably forever. Do not store in polypropylene tubes.
Polystyrene or PET are better. Be sure to mix well when you thaw.
2. Some researchers use polyethyleneimine instead of
polylysine. Are there advantages of one over the other?
PEI is cheaper than polylysine. We have not had much
success with PEI, but Mark Mattson uses it all the
time, after starting culture in Neurobasal/10% serum.
Reports suggest no significant differences in neuron
survival or spike rates.
Soussou WV, Yoon GJ, Brinton RD, Berger TW (2007) Neuronal network
morphology and electrophysiologyof hippocampal neurons cultured on
surface-treated multielectrode arrays. IEEE Trans Biomed Eng
54:1309-1320. PM:17605362
Brewer GJ, Cotman CW (1989) Survival and growth of hippocampal neurons
in defined medium at low density: advantages of a sandwich culture
technique or low oxygen. Brain Res 494:65-74. PM:2765923
3. BrainBits neurons are growing more as clusters or
clumps of cells instead of isolated. What's wrong?
This means that adhesion of cells to themselves is tighter than
to the substrate. The most likely problem is a poorly prepared
substrate. Compare your protocol to the recommended
preparation of polylysine and coating of substrates. Protocol
4. Regarding poly-L-lysine vs poly-D-lysine, I assume that
poly-D-lysine is used because if it breaks down to lysine,
the Dlysine can't be used by cells. Any truth to that?
That's the theory. In some early studies (Brain Res. 494:65
(1989)) we found little difference.
BrainBits Tissue Back to top
1. I assume the BrainBits come from newborn
animals?
They come from embryonic day 18 rats unless otherwise specified.
2. Cortical cultures do not include hippocampus,
right?
They could, but we prepare E18 cortex without thalamus or
hippocampus. We can provide either hippocampus tissue or
cortex tissue by express mail. These "BrainBits" allow
preparation of cells in 20 minutes for $109 for >1 million
hippocampal neurons or 2 million cortical neurons.
3. How many neurons can come
with each vial from cortex and hippocampus?
The number of neuons for hippocampal neurons is >1 million
or 2 million for cortical neurons.
4. When do hippocampal neurons show excitotoxicity?
Acetylcholine is not toxic at any age that we know of.
Glutamate is maximally toxic starting at 7 days of
culture from E18 hippocampus. It may be 1 or 2 days
earlier for cortical neurons.
5. Do you know how many astrocytes you provide
with each vial from cortex and hippocampus?
The number of astrocytes to start from hippocampus
is >1 million, cortex >2million.
6. What assurances are there that animal brain and spinal
cord tissue is aquired under an NIH approved protocol?
BrainBits animal protocol #32-08-013 was approved by
the Southern Illinois University School of Medicine
Laboratory Animal Care and Use Committee on 6/04/2008
For NIH grants, vertebrate animals section:
F. Vertebrate animals
1. Hippocampal, cortical, and other brain and spinal cord
neurons for primary culture will be obtained from the brains
of embryonic or early postnatal rats or mice. Brains will be
dissected after anesthesia with halothane (mice) or IP
pentabarbital (rats).
2. By isolating neurons from animals, many more experimental
variables can be studied than in whole animal studies, thus
reducing the need for animals. Rat and mouse brains are the
best studied of all animal brains.
3. The average housing needs are estimated at 2 days, enough
for the rodents to recover from the trauma of transportation.
Animals will be cared for by SIUSM Laboratory Animal Medicine,
which is accredited by the AALAC and has a staff of 10 under
the direction of Teresa Liberati, D.V.M., Ph.D., certified by the
ACLAM.
4. Brains will be dissected after anesthesia with halothane or
pentabarbital. Thus, these procedures limit use of animals
to that which is unaboidable in the conduct of scientifically
valuable research and minimize discomfort, distress and pain
to the animals.
5. Brains will be dissected after anesthesia with halothane.
Pentabarbital will be administered to the heart for rats. Mice
will be guillotined under anesthesia. These methods of
of euthanasia are consistent with the recommendations of the
Panel on euthanasia of the American Veterinary Medical
Association.
7. Can BrainBits, LLC aquire dissected regions from the pregnant
mothers of our embryonic rats and mice?
BrainBits could supply brain cortices from pregnant mothers of our
embryonic rats or mice. Here is a suggested protocol for you to
evaluate whether you want to try this. Please tell us if and when you
want BrainBits to begin sending you adult cortices for your isolation
of endothelial cells.
Wolburg H, Neuhaus J, Kniesel U, Krauss B, Schmid EM,
Ocalan M, Farrell C, Risau W (1994) Modulation of tight junction
structure in blood-brain barrier endothelial cells. Effects of tissue culture,
second messengers and cocultured astrocytes.
J Cell Sci 107 ( Pt 5):1347-1357. PM:1692329
Risau W, Engelhardt B, Wekerle H (1990) Immune function of the
blood-brain barrier: incomplete presentation of protein (auto-)antigens
by rat brain microvascular endothelium in vitro.
J Cell Biol 110:1757-1766. PM:7929640
8. More information on isolation of motor neurons from spinal cord
M. Das, J. W. Rumsey, N. Bhargava, M. Stancescu, and J. J. Hickman. A defined
long-term in vitro tissue engineered model of neuromuscular junctions. Biomaterials
1. Is there a problem with triturating the tissue through
a 18 or 20 gauge needle on a tuberculin syringe?
Yes. The edge of the needle is too sharp. The blue plastic
tip of a 1 ml pipet is better, but the best is a flame polished 9
inch pasteur pipet that was previously silanized. Be sure that
tip opening is at least 1mm diameter.
2. Does hippocampal or cortical cell viability depend on
the method of dissociation? Do you prefer mechanical
or enzymatic dissociation?
Yes, higher viable yield is obtained with papain
(see J Neurosci Meth 71:143) but digestion of surface proteins
is inevitable. For short term (less than 4 days) this is a
concern. For longer term, it probably is not significant.
The higher viability is obtained with a fire polished 9 inch glass
pipet, lower with a blue plastic pipet tip.
3. Do you use enzymes to prepare cortical cultures?
For E18, we use mechanical for speed and simplicity. For
higher yields, we use Worthington papain at 2mg/ml. See Protocol
4. Why do you recommend Hibernate-Ca without B27 for
optimum dissociation with papain?
Calcium promotes adhesion. Therefore, removing
calcium promotes the dissociation of neurons.
To offer cell adheasion proteins as the primary
substrate for papain, B27 is ommitted so that
proteins in B27 don't compete for substrate.
Cultures Back to top
1. For how many passages and for how long will the
cells survive?
If you feed the cells with one-half medium volume change
every 3-4 days, they will last for months. Since they do
not multiply, it does not make sense to passage them.
If you add FGF2 (bFGF) at 5 ng/ml, then they will multiply
as long as you keep the density below about 240
cells/mm2.
2. How many astrocytes does it contain?
There are less than 5% astrocytes in the hippocampal
BrainBits. The cortical BrainBits probably has about
10% astrocytes. If you culture in serum, you will get
many more. We recommend culture in B27/Neurobasal
(Invitrogen/GIBCO). This inhibits glial growth without
AraC.
3. When should I add Ara-C as a mitotic inhibitor
for astroglia?
In the Neurobasal/B27 culture medium that we recommend,
you do not need to add Ara-C at all. The medium does not
artificially stimulate astroglial proliferation like serum does.
Try it!
4. My cultures started with BrainBits tissue are
contaminated. They looked fine at the start,
but now the medium is cloudy and yellow with
numerous 1-3 um long phase dark bodies.
There are at least 5 possible sources of
contamination (assuming other cultures
in your incubator are not contaminated and the
humidifying water in the base of the incubator
is clear):
1. The culture vessel itself. Control by adding
freshly filtered medium to a number of culture
vessels.
2. The adhesive substrate material. This is not
uncommon. BrainBits has prepared sterile
substrate cover slips for you to test. You can
also put freshly filtered culture medium on your
prepared substrates to look for this source of
contamination.
3. The medium itself or one of its additives
(e.g., glutamine). This is not uncommon. Be sure
to include a separate test in an uncoated well of
your medium and one supplied by BrainBits.
4. Seemingly random or low levels of contamination
can come from talking or even breathing in the
direction of an open culture. Also, the air between
the sterile hood and the incubator is not sterile and
most culture vessels are not sealed. If there is a long
distance from the hood to the incubator, you can
place your cultures in a plastic food container that has
been rinsed with 70% ethanol.
5 The BrainBits tissue. Although we perform sterility
tests on the media that we supply, the tissue itself
can not be tested. If you can provide evidence that
your BrainBits tissue arrived on time and was stored
at 4 degrees C until use and the above 4 possibilities
have been ruled out, please call us for a one time
replacement. Usually, our experience is that yours is
the only sample contaminated out of all our shipments
for the week, so please understand that we require the
above tests.
5. I was wondering if you could give me advice on how many
cells I would need to plate in order to get enough protein for
several western blots, i.e. how many cells would equal
20-50 ug of protein? Also, for how many weeks will they
continue to split so I can have multiple cultures, and then how
long will they survive after they no longer divide?
According to Patel and Brewer (2003), each 18,000 cells grown
for a week in culture has about 6 ug protein. So, 3000 cells/ ug
protein. A western blot extract that needs about 50 ug protein
would require about 15,000 cells.
Frozen Cells Back to top
1. I tried a viability stain with a 1:1 dilution of trypan blue from freshly
thawed frozen neurons . All the cells seem to stain light blue.
This is to be expected since the cryoprotectant partially permeabilizes
the membrane. Handle these freshly thawed cells very gently. After
several hours in culture or certainly after 15 hr., compared to competitors
frozen cells, a larger number of these cells will completely exclude trypan
blue as an indication of viability. You could also try staining for dead cells
with the red fluorescent dye propidium iodide at 5 ug/mL.
NbActiv4® Back to top
1. What can you tell me about the improvements in growth conditions and also what supplements I would need (ex. glutamine, etc. ) to plate and grow primary E18 cortical neurons with this new media?
You can use the new medium just like you would normally use the mixture of Neurobasal/B27 and 0.5 mM Glutamax, because all these ingredients are in the new mix+ 3 new ones cholesterol, estrogen and creatine.
Precoated Culture Flasks Back to top
1. Can you recommend any brand of pre-coated
Tissue culture flask or method of preparing tissue
culture flasks for growth of primary neurons?
I would like to infect cells to up regulate a gene of
interest and then harvest all the cells at once to
perform WB and IP experiments.
BrainBits has determined that poly-lysine coating
on cell culture plastic or glass measurably begins to
lose effectiveness within 2 days of coating. We
suspect that some pre-coated plates work
satisfactorily, but our experience is that a number of
them result in poor adhesion and survival of primary
neurons. Therefore, we recommend that you coat your
own substrates and use them within a day, as described
in the protocol provided.
Progenitors Back to top
1. Why do I see clusters forming right away?
Seeding cells at 30,000/cm2 will result in rapid cluster
formation. Without attachment, they will stay as progenitors
in Neuropro and grow into neurospheres that can be
harvested in 4-7 days. Evaluation with immunostaining for
nestin or other antibodies can begin after 4 days. To
see clonal growth, you can plate the original sample or these
neurospheres at limiting dilution of 50 cells/cm2 of tissue
culture plastic or ultra-low adhesion plastic in the same medium.
To see pluripotency, plate dissociated neurospheres at
5 to 15 thousand cells/cm2 on substrates coated with
poly-d-lysine (50 ug/ml water).
2. Should I seed single cells to do the neurosphere assay?
No, a neurosphere contains a cluster of cells. You can fix
them and stain as a cluster or dissociate them with papain
or trypsin, count to determine yield and/or fix to evaluate
individual cells.
3. The cells were single after seeding but they formed
cluster today. The sizes of some of the clusters
are very big. Is this the right sign?
Yes.
4. Should I observe the neurosphere formation to evaluate
the cells' progenitor stage
No, wait at least 4 days.
5. How do I distinguish the neurosphere and cluster?
You can pass the neurospheres onto low adhesion plastic
or onto adhesive surface and see differentiation. As long
as cells are on low-adhesion plastic, they will stay as
progenitors.
6. Do I need to do immunostaining at this stage?
Wait at least 4 days.
Ordering Back to top
1. How do I write a purchase order (including F.E.I.N.)?
In order of preference: E-mail, Webstore, FAX , or mail your
Purchase Order (PO) number to BrainBits. Please provide
billing and shipping information, desired quantities and shipping
dates to BrainBits at the numbers below. The FEIN for
BrainBits is 20-0023331.
2. Are they available any time, or there is a kind of
schedule/delay for the shipment?
Usually within one days notice.
3. Do you know an agency YASHIMA (or WAKO), chemical
supplier, in Japan?
BrainBits has good experience with overseas shipping. Most
important is to provide a customs declaration page and make
arrangements for clearance through customs. There is a $30
surcharge for international orders. This fee is waived if you
pay by credit card though either FAX or Email. International
orders require payment in advance.
4. What are the fees for wire transfers?
Any and all wire transfer fees incurred by all banks are the
customer's responsibility to pay. Please contact us prior to
payment for our bank account information and transfer fee
amounts.
Other Back to top
1. Should I be aware of any ethical issues?
Use of Hibernate E for human embryonic tissue or human
embryonic stem cells is considered unethical and is forbidden.
Hibernate Use Form
Current procedures are approved by an Institutional Animal Use and Care Committee.
Transfection Back to top
Information on transiently transfecting neurons.
Lakkaraju A, Dubinsky JM, Low WC, Rahman YE (2001) Neurons Are Protected from Excitotoxic Death by p53 Antisense Oligonuceotides Delivered in Anionic Liposomes. J Biol Chem 276: 32000-32007.JBC:32000. JBC:32000
Ohki, E. C., Tilkins, M. L., Ciccarone, V. C., and Price, P. J. Improving the transfection efficiency of post-mitotic neurons. J Neurosci Methods 112, 95-99. 2001. PM:11716945
Grigston, J. C., Vandongen, H. M., McNamara Ii, J. O., and Vandongen, A. M. Translation of an integral membrane protein in distal dendrites of hippocampal neurons. Eur J Neurosci 21, 1457-1468. 2005. PM:15845074
McNamara, J. O., Grigston, J. C., Vandongen, H. M., and Vandongen, A. M. Rapid dendritic transport of TGN38, a putative cargo receptor. Brain Res Mol.Brain Res 127, 68-78. 2004. PM:15306122
*Hibernate, Neurobasal and B27 is for research purposes only and is manufactured by Invitrogen Corporation. Hibernate, Neurobasal, and B27 is a trademark of Invitrogen Corporation.