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 6 months.
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 us as a complete, ready to use formulation NbActv1 and separately through Invitrogen.
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 µm 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 370C in ambient CO2, the LD50 in Neurobasal/B27/gln is24 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 Hibernate include:
Brain storage during genotyping of transgenic mice
Shipping brain tissue to collaborators
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.
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 d lysine 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.
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 $136 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 5/18/11.
For NIH grants, vertebrate animals section:
Assurance Number is A-3209-01
F. Vertebrate animals
- 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 pentabarbitol (rats).
- 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.
- 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.
- Brains will be dissected after anesthesia with halothane or pentobarbital. 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.
- 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 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
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 31 (18):4880-4888, 2010. PM:20346499
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. For E18 tissue, we use mechanical for speed and simplicity.
3. Do you use enzymes to prepare cortical cultures?
For higher yields, we use a 2 mg/ml solution of papain diluted in Hibernate E-Ca without B27. This solution should be incubated in a 30oC water bath for 10 minutes. Additional chemicals such as EDTA, Mercaptoethanol, or Cysteine-HCl should NOT be added to this solution. Please refer to our Primary Neuron Cell Culture Protocol for addditional information.
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 adhesion proteins as the primary substrate for papain, B27 is omitted so that proteins in B27 don't compete for substrate.
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.
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):
- The culture vessel itself. Control by adding freshly filtered medium to a number of culture vessels.
- 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.
- 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.
- 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.
- 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?
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 150,000 cells.
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.
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.
Pre-Coated Culture Flasks
1. Can you recommend any brand of pre-coated Tissue culture flask or method of preparing tissue culture flasks for growth of primary neurons?
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.
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?
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.
1. How do I place a purchase order?
In order of preference: Webstore, E-mail, FAX , or mail your Purchase Order (PO) to BrainBits. Please include your billing and shipping information, desired quantities and shipping dates. Our contact information is below:
BrainBits LLC, 510 Apple Orchard Rd, Suite 100, Springfield, IL 62703
Toll Free: 877-201-3057 * Tel: 217-789-9313 * FAX: 217-789-9314
2. Payment Terms
BrainBits LLC payment terms are Net 30.
3. Are they available any time, or there is a kind of schedule/delay for the shipment?
Usually within one days notice. Special orders can take from 1 - 4 weeks for arrival.
4. Do you know an agency YASHIMA (or WAKO), chemical supplier, in Japan?
BrainBits LLC provides shipments to Puerto Rico, Canada, and Mexico. BrainBits UK 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 surcharge for international orders (outside the EU) paid via wire transfer. This fee is waived if you pay by credit card though either FAX or Email. International orders (outside the EU) require payment in advance. Please view our BrainBits UK website at www.BrainBits.co.uk.
5. 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.
1. Should I be aware of any ethical issues?
Current procedures are approved by an Institutional Animal Use and Care Committee.
Transfecting BrainBits® Neurons
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 BiolChem 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 Methods112, 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.