Culture Media
1. How long is the Culture Media (Neurobasal®/B27®/GlutaMAX™) stable?
Our culture media is stored at 4oC in the dark for up to 6 months. To prevent oxidation, media can be aliquoted into smaller volumes and stored at 4oC.
2. How often do I need to do a media exchange?
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. What is the difference between NbActiv1™ and NbActiv4®?
NbActiv4® contains the same ingredients as NbActiv1™ plus Cholesterol, Estrogen, and Creatine. NbActiv4® is used mainly as electrophysiology media.
4. When plating Hippocampal cultures, you typically add 25uM glutamate to the NbActiv1™ (Neurobasal®/B27®/GlutaMAX™) or NbActiv4® (Neurobasal®/B27®/GlutaMAX
™/Cholesterol/Estrogen/Creatine) up to the 4th day in culture. What is the purpose of the glutamate?
It helps the hippocampal neurons sprout and promotes viability. After DIV4 a half media exchange should be done with media that does not contain glutamate.
5. Is there a problem if glutamate is left out?
You will get about 30% lower survival at DIV4.
6. If I plate the cells in NbActiv1™ can I change the media to NbActiv4®?
Yes, at any time in culture, the media can be changed from NbAcitv1™ to NbActiv4®. However, the opposite is not true. Cells plated in NbActiv4® should not be changed to NbActiv1™.
7. What additional supplies do I need to maintain the cultures?
Each 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 and separately through Invitrogen. You will also need coated glass or plastic substrates of your choosing, we recommend poly-D-lysine for substrate. As well as this, you will need dissociation solution, we recommend our Papain and Hibernate® E-Ca and a fire polished pipette. We include all these supplies in our Complete Culture Kit.
8. Should I be supplementing the plating medium with antibiotics/antimycotics? e.g.: pen/strep/fungizone or gentamicin?
We usually culture without antimicrobial antibiotics and antimycotics 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. The use of antibiotics is a common practice, and the type and concentration are up to the end user.
Hibernate®, Neurobasal® and B27® are for research purposes only and are manufactured by Invitrogen Corporation. Hibernate®, Neurobasal® and B27® are a trademark of Invitrogen Corporation.
1. How long is the Hibernate® Media stable?
Our Hibernate is stored at 4oC in the dark for up to 1 year. To prevent oxidation, media can be aliquoted into smaller volumes and stored at 4oC.
2. Do you always add B27®, and GlutaMAX™ to Hibernate®, or does it keep the cells alive in just Hibernate®?
Almost always. Please see Figure 1 of Brewer and Price (1996) PM:8856709 At 37oC in ambient CO2, the LD50 in Neurobasal®/B27®/GlutaMAX™ is 24 hours, and in Hibernate®/B27®/GlutaMAX® it is greater than 3 days.
3. Can we supply custom formulation of Hibernate?
Yes, we can supply custom formulation of Hibernate.
4. Other uses of Hibernate include:
Brain storage during genotyping of transgenic mice.
Shipping brain tissue to collaborators.
1. My neurons are growing more as clusters or clumps of cells instead of isolated. What is 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 poly-D-lysine and coating of substrates. Another possibility is the plating density may be too high.
2. 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 cannot be used by cells. Any truth to that?
That is the theory. In some early studies (Brain Res. 494:65(1989)) we found little difference.
3. Some researchers use polyethyleneimine (PEI) instead of poly-D-lysine (PDL). Are there advantages of one over the other?
PEI is cheaper than PDL. 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 electrophysiology of 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.
Pre-coated PDL Cultureware
1. Can you recommend any brand of pre-coated tissue culture flask or method of preparing tissue culture flasks for growth of primary neurons?
We have determined that poly-D-lysine coating on cell culture plastic or glass measurably begins to lose effectiveness after coating.Coated glass slips lose effectiveness after 14 days, and coated plastic after 10 days. We suspect that some pre-coated plates work satisfactorily, but our experience is that several result in poor adhesion and survival of primary neurons. Therefore, we recommend that you coat your own substrates and use them within a week or following the days above.
2. Do you sell pre-coated cultureware?
Yes, we sell PDL coated Flasks, Well Plates and Slips. We coat these overnight and ship them the next day for overnight delivery. We recommend that you use these within one week.
Fresh Tissue
1. What age tissues do you offer?
Our standard tissue is embryonic day 18 or adult tissue from the pregnant moms (Ages 3-4 months). All other tissue ages are available for special order with a lead time of 2-3 weeks.
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
2. How many neurons do you guarantee for each vial from cortex and hippocampus?
The number of neurons guaranteed is 1 million for Hippocampus or 2 million for Cortical neurons. Numbers differ for other tissue types.
3. What assurances are there that animal brain and spinal cord tissue is acquired under an NIH approved protocol?
Our animal protocol #233-08-013 was approved by the Southern Illinois University School of Medicine Laboratory Animal Care and Use Committee on 04/07/2020.
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. Pentobarbital will be administered to the heart for rats. Mice will be guillotined or cervical break under anesthesia. These methods of euthanasia are consistent with the recommendations of the Panel on euthanasia of the American Veterinary Medical Association.
- 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 Helen Valentine, D.V.M., MS, certified by the DACLAM
- Brains will be dissected after anesthesia with halothane. Pentobarbital will be administered to the heart for rats. Mice will be guillotined or cervical break under anesthesia. These methods of euthanasia are consistent with the recommendations of the Panel on euthanasia of the American Veterinary Medical Association. Thus, these procedures limit use of animals to that which is unavoidable in the conduct of scientifically valuable research and minimize discomfort, distress, and pain to the animals.
Frozen Cells
1. Do you offer Frozen Cell lines?
Currently, we do not offer frozen cells and do not have a protocol for freezing cells. You can request flash freezing as a custom option for an additional processing fee.
Dissociation
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, and lower visibility with a blue plastic pipette 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 2mg/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 additional information.
4. Why do you recommend Hibernate® E-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® do not compete for substrate.
1. Do primary cells proliferate and if so, how many times can you passage them?
Primary cells do not proliferate and should not be passaged. If passaged only 30% of cells survive and cells cannot be passaged a second time.
2. Do you know how many astrocytes you provide with each vial of tissue?
The number of Astrocytes is 20% of the cell number. When growing Neuronal cultures, a media without Serum (NbActiv1™) is used to grow cultures with less than 2% Astrocytes. This inhibits glial growth without AraC. When growing Astrocytes, culture medium with Serum (NbAstro™ - Neurobasal®/Horse Serum/GlutaMAX™) can be used to bring Astrocytes to confluence in 1.5 to 2 weeks.
3. When should I add AraC as a mitotic inhibitor for astroglia?
In the Neurobasal®/B27®/GlutaMAX™ culture medium that we recommend, you do not need to add AraC at all. The medium does not artificially stimulate Astroglia proliferation, like serum does.
4. Do Astrocytes proliferate and if so, how many times can they be passaged?
Astrocytes do proliferate and can be passaged. Our E18 Astrocytes can be passaged successfully up to 4-5 times.
5. 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.
6. My cultures 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 could be the source of contamination. To rule this out add freshly filtered medium to another culture vessels. If the media turns yellow or cloudy the vessel is contaminated.
- The adhesive substrate material. This is common. You can 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., glutamate). This is common. Be sure to include a separate test in an uncoated well of your medium and one supplied by us.
- 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 tissue. Although we perform sterility tests on the media that we supply, the tissue itself cannot be tested. If you can provide evidence that your tissue arrived on time and was stored at 4o 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.
7. I was wondering if you could give me advice on how many cells, I would need to plate 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 g protein would require about 150,000 cells.
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Transnetyx Tissue standard payment terms are Net 30.
3. Do you offer any multi-unit discounts?
Yes, some of our catalogue has multi-unit discounts. To receive the discount, the items must ship at the same time.
4. What is your shipping schedule?
We can ship most items within 24 hours of receiving the order. E 18 and Adult Rat and Mouse tissue is shipped on Tuesdays, Wednesdays, and Thursdays. Media and additional supplies ship Mondays through Thursdays. For Custom or Special-Order items, we require 2-3 weeks lead time for the order.
5. Do you ship internationally?
Transnetyx Tissue provides shipments Internationally via FedEx. If your company has a FedEx account they would like to charge for the freight and duties & taxes cost, please indicate that on your order. Otherwise, freight and duties & taxes will be included on your invoice.
All International shipments are scheduled on Mondays for Media or Tuesdays for Tissue. This allows for clearance time through customs. Please submit orders at least one business day in advance to accommodate this shipping schedule. Please make sure to submit any customs documentation that is specific to your country. We only provide standard customs documentation and cannot be held responsible for delays in customs due to insufficient paperwork.
1. Should I be aware of any ethical issues?
Current procedures are approved by an Institutional Animal Use and Care Committee.