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NeuroPure™ FAQ's

Frequently asked questions: NeuroPure™ Primary Rat Neuronal Cells

1. What are the NeuroPure Primary Rat Neuronal Cells? The NeuroPure Primary Rat Neuronal Cells are live neurons isolated from micro-surgically dissected regions of day 18 embryonic Sprague/Dawley or Fischer 344 rat brain. These cells are prepared fresh each week and shipped in a nutrient rich medium that keeps the cells alive for up to 14 days under refrigeration.

2. For what applications can the NeuroPure cells be used? NeuroPure cells can be used for a wide variety of applications including: plasmid and siRNA transfections, neuron development studies, assessment of molecular pharmacology and toxicology, neuronal physiology studies, assessment of electrophysiological properties, stroke studies, drug screening, immunoreactivity studies, Alzheimer’s Disease studies, immunocytochemistry, schizophrenia studies, patch clamping, studies of glutamate-induced neuronal degeneration, and measurement of intracellular calcium and other ions, etc.

3. How long will the NeuroPure cells survive in culture? Can they be passed? If you feed the cells with a ½ volume medium change every 3-4 days, they will last for months. Since they don’t multiply, it doesn’t 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 ~240 cells/mm2.

4. How many astrocytes do the NeuroPure neuronal cells contain? There are <5% astrocytes in the NeuroPure hippocampal cells. The NeuroPure cortical cells typically contain <10% astrocytes. However, if you culture the cells in serum-containing medium, you will get many more astrocytes. We recommend culturing in B27/Neurobasal medium. This inhibits glial growth without Ara_C.

5. How do I order NeuroPure Primary Rat Neuronal Cells? You may place your order via phone (888-428-0558), fax (858-623-9494), email (orders@genlantis.com), or website (www.genlantis.com). Orders taken Monday to Friday are shipped the following Wednesday for overnight delivery.

6. Do you recommend mechanical or enzymatic dissociation for the NeuroPure cells? We recommend mechanical dissociation. Specifically, we suggest triturating the cells ~10 times (in about 30 seconds) using a siliconized 9-inch pasteur pipette with the tip barely fire polished to an opening of 0.7–0.9 mm D (best results) or with a 1 ml blue polypropylene pipette tip with an opening of 0.9 mm D.

7. Can enzymatic dissociation methods be used for the NeuroPure™ cells? Yes. Higher viable yields are obtained with papain (as opposed to trypsin). Please note that some digestion of surface proteins is inevitable when using enzymatic dissociation. For short term (less than 4 days) this is a concern. For longer term, it probably is not significant. The highest viability is obtained by mechanical dissociation using a fire polished 9 inch glass pipette, lower with a blue plastic pipette tip.

8. Do NeuroPure cells express glutamate receptors? NeuroPure cells, having been derived from embryonic rats, have not yet expressed their glutamate receptors. However, they will express glutamate receptors after approximately 1 week in culture.

9. Once I prepare the B27 supplement / Neurobasal media / 0.5 mM glutamine, how long is the media stable? We store the medium at 4 ºC in the dark for up to 1 week.

10. 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.

11. What is the purpose of the glutamate contained in the plating media provided with the NeuroPure cells? It helps the cells sprout and promotes viability.

12. Do I need to supply media or reagents to maintain the NeuroPure cells? You will need to supply coated glass or plastic substrates of your choosing. The cells are provided with enough medium to start cultures and maintain them for 4 or 5 days. Beyond that, you will need more culture medium. We recommend Neurobasal with B27 + 0.5 mM glutamine.

13. What poly-D-lysine do you use to coat your plates? Sigma P6407, poly-D-lysine hydrobromide, 5 mg.

14. I do not 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 NeuroPure™ cells? Probably forever. However, do not store in polypropylene tubes; polystyrene is better. Be sure to mix well when you thaw.

15. Some researchers use polyethyleneimine instead of polylysine. Are there advantages of one over the other? PEI is cheaper than polylysine. However, we have not had much success with PEI. Therefore, we recommend polylysine.

16. The NeuroPure™ neurons are growing more as clusters or clumps of cells instead of isolated cells. 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.

17. Do the NeuroPure cortical cells include any hippocampal cells? There could be a very small percentage. However, we prepare the cortex without thalamus or hippocampus.

18. Is there a problem with triturating the tissue through an 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 9-inch pasteur pipette with the tip barely fire polished to an opening of 0.7–0.9 mm D.

19. Should I be aware of any ethical issues? Current procedures for preparing the NeuroPure cells are approved by an Institutional Animal Use and Care Committee.

20. Your protocol recommends to incubate the cortical cells at 37oC with 5% C02 and/or 9% or 20% oxygen. Which is best? 9% oxygen is more physiological for the brain. Only lungs and epidermis are typically exposed to 20% oxygen. However, Most CO2 incubators set for 5% CO2 will fill the balance with room air, which will be 20% oxygen. This is fine for NeuroPure cells.


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Frequently asked questions: NeuroPure™ Primary Rat Neuronal Cells

1. What are the NeuroPure Primary Rat Neuronal Cells? The NeuroPure Primary Rat Neuronal Cells are live neurons isolated from micro-surgically dissected regions of day 18 embryonic Sprague/Dawley or Fischer 344 rat brain. These cells are prepared fresh each week and shipped in a nutrient rich medium that keeps the cells alive for up to 14 days under refrigeration.



2. For what applications can the NeuroPure cells be used? NeuroPure cells can be used for a wide variety of applications including: plasmid and siRNA transfections, neuron development studies, assessment of molecular pharmacology and toxicology, neuronal physiology studies, assessment of electrophysiological properties, stroke studies, drug screening, immunoreactivity studies, Alzheimer’s Disease studies, immunocytochemistry, schizophrenia studies, patch clamping, studies of glutamate-induced neuronal degeneration, and measurement of intracellular calcium and other ions, etc.



3. How long will the NeuroPure cells survive in culture? Can they be passed? If you feed the cells with a ½ volume medium change every 3-4 days, they will last for months. Since they don’t multiply, it doesn’t 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 ~240 cells/mm2.



4. How many astrocytes do the NeuroPure neuronal cells contain? There are <5% astrocytes in the NeuroPure hippocampal cells. The NeuroPure cortical cells typically contain <10% astrocytes. However, if you culture the cells in serum-containing medium, you will get many more astrocytes. We recommend culturing in B27/Neurobasal medium. This inhibits glial growth without Ara_C.



5. How do I order NeuroPure Primary Rat Neuronal Cells? You may place your order via phone (888-428-0558), fax (858-623-9494), email (orders@genlantis.com), or website (www.genlantis.com). Orders taken Monday to Friday are shipped the following Wednesday for overnight delivery.



6. Do you recommend mechanical or enzymatic dissociation for the NeuroPure cells? We recommend mechanical dissociation. Specifically, we suggest triturating the cells ~10 times (in about 30 seconds) using a siliconized 9-inch pasteur pipette with the tip barely fire polished to an opening of 0.7–0.9 mm D (best results) or with a 1 ml blue polypropylene pipette tip with an opening of 0.9 mm D.



7. Can enzymatic dissociation methods be used for the NeuroPure™ cells? Yes. Higher viable yields are obtained with papain (as opposed to trypsin). Please note that some digestion of surface proteins is inevitable when using enzymatic dissociation. For short term (less than 4 days) this is a concern. For longer term, it probably is not significant. The highest viability is obtained by mechanical dissociation using a fire polished 9 inch glass pipette, lower with a blue plastic pipette tip.



8. Do NeuroPure cells express glutamate receptors? NeuroPure cells, having been derived from embryonic rats, have not yet expressed their glutamate receptors. However, they will express glutamate receptors after approximately 1 week in culture.



9. Once I prepare the B27 supplement / Neurobasal media / 0.5 mM glutamine, how long is the media stable? We store the medium at 4 ºC in the dark for up to 1 week.



10. 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.



11. What is the purpose of the glutamate contained in the plating media provided with the NeuroPure cells? It helps the cells sprout and promotes viability.



12. Do I need to supply media or reagents to maintain the NeuroPure cells? You will need to supply coated glass or plastic substrates of your choosing. The cells are provided with enough medium to start cultures and maintain them for 4 or 5 days. Beyond that, you will need more culture medium. We recommend Neurobasal with B27 + 0.5 mM glutamine.



13. What poly-D-lysine do you use to coat your plates? Sigma P6407, poly-D-lysine hydrobromide, 5 mg.



14. I do not 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 NeuroPure™ cells? Probably forever. However, do not store in polypropylene tubes; polystyrene is better. Be sure to mix well when you thaw.



15. Some researchers use polyethyleneimine instead of polylysine. Are there advantages of one over the other? PEI is cheaper than polylysine. However, we have not had much success with PEI. Therefore, we recommend polylysine.



16. The NeuroPure™ neurons are growing more as clusters or clumps of cells instead of isolated cells. 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.



17. Do the NeuroPure cortical cells include any hippocampal cells? There could be a very small percentage. However, we prepare the cortex without thalamus or hippocampus.



18. Is there a problem with triturating the tissue through an 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 9-inch pasteur pipette with the tip barely fire polished to an opening of 0.7–0.9 mm D.



19. Should I be aware of any ethical issues? Current procedures for preparing the NeuroPure cells are approved by an Institutional Animal Use and Care Committee.



20. Your protocol recommends to incubate the cortical cells at 37oC with 5% C02 and/or 9% or 20% oxygen. Which is best? 9% oxygen is more physiological for the brain. Only lungs and epidermis are typically exposed to 20% oxygen. However, Most CO2 incubators set for 5% CO2 will fill the balance with room air, which will be 20% oxygen. This is fine for NeuroPure cells.

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