Science method

Neuron Culture - Science method

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Have you noticed any changes recently with cell culture using N2 or B27-supplements ? We have an anormal cell death in our retinal explants lately and we checked other parameters (media not expired, incubators OK, no contamination including no mycoplasma contamination...). Thanks for your input.
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Yes, we are also cultivating neuroprogenitor cells and neurons and have recently encountered a similar issue. Unfortunately, We observed significant cell loss, aand even when we attempted to expand them, they do not survive. Like you, we verified key parameters, but found no abnormalities. Interestingly, other cell types that don't use B27 and N2 do not seem to exhibit this problem.
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I'm patching neurons (mouse hippocampus) in neuronal culture coverslips. Experiment design: enter whole-cell mode, fill the neurons with dyes for 5-20 minutes, withdraw the pipette, perform imaging experiments.
I am able to do this but I would like to increase the success rate.
When I mechanically withdraw the pipette (45 degree angle, slowly) sometimes large chunks of the cell body come with it, resulting in cell death.
I have been using 2-6 MOhm resistance pipettes, no obvious relationship between resistance and success rate in my hands. I have found that polishing pipettes seems to help.
Any tips welcome.
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Great, thank you very much Martijn Sierksma !
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Hello guys!
So, Its been 4 years that I do primary cortical neuronal culture as the same way and they are cultived for 12 days at plates covered by Poly D lisine and with the medium: Neurobasal + B27 + Glutamax + Gentamicin. In the past month, some of my neurons didnt developed! Actually, the cells stayed at progenitors cells (the same as they looked like at day 1). The cells are not dead, as I noticed at microscope and by medium color changes, but they dont develop to neurons. Any ideas of why this could be happening? The incubator is the same as usual, and I share with another researcher that cultivates astrocytes and they are just fine!
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Dear Amanda,
Here few things to try in your next culture:
> Avoid Gentamicin >> check what happens
> Try replacing Neurobasal by DMEM in your culture media (with and without Gentamicin
> Check for mycoplasma contamination either in culture or you animals (more if you breed them yourself).
Dirty troubleshooting :
> What age are the embryos pups you use? Assuming you use a Banker's style protocol, If using E16-E19 embryos, try leaving them longer (4-6hs some use overnight) in media with serum to allow more astrocytes to attach (dirtier culture). This is so after change to neuronal media, you will have many astrocytes to condition de media. If under this condition you see your cells differentiating better than in your regular cultures, then you will know that something in your original media formulation isn't working properly, and your neurons are not getting all the goodies they need. You can also add Gentamicin in this condition to test whether it could be the cause of your problems. I have never used any antibiotic for primary neuronal cultures outside for selection and for short periods of time.
Happy to clarify is something of the above doesn't make sense to you.
Best and good luck
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Hello, I am Trisha. I am currently trying to isolate lipids from primary cortical neuronal cultures. I want to do the Total phosphatidic acid assay using Cellbiolabs-MET5019 kit and also following the company´s protocol for lipid extraction. My samples when liquid looks clear, and then I vacuum dry it in order to dilute with the Assay buffer. But, after drying, I am getting a greyish pellet which is difficult to dissolve in the assay buffer. Therefore, it would be grateful if someone has experience in this matter and help me with suggestions. Thank you in advance.
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Have you tried contacting the company? Each company's kits are generally different from each other. Having said that try sonicating in a sonicating bath. You will have extracted things, not all of which will be resolubilized in the buffer. That's ok. Sonication should give you a cloudy solution as it will resuspend everything there. Spin in a tabletop centrifuge at max speed for a couple minutes to pull down the insoluble materials and use the (hopefully) clear supernatant in the assay.
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Hello everyone,
I'm a phD student doing primary cortical neurons culture for 3 years. I'm doing this protocol from embryonic mice (E16) and following these steps
-Dissection in cold HBSS 1X +mg +Ca
-Cortices are kept in DMEM high glucose + glutamax supplemented with 10% FBS and 1% P/S
- After DMEM complemented is removed and trypsin 0.05% is add and cortices are incubated for 15 min at 37°C.
- Trypsin is removed and cortices are washed with DMEM complemented to stop the reaction, then dissociated in 1 mL with a P1000 (10-15 up and down) and filtered with a 40µM cell filter.
- Then we add 9mL of DMEM complemented to dilute cells and we count.
- Cells are plated in precoated PLL plates (100mg/L, washed 3 times with H2O) with DMEM complemented, 37°C 5% CO2.
- After 4 hours, medium is fully changed with Neurobasal + B27+ gentamycin + Glutamax
This protocol was working really well in our hands but since this summer is not working anymore.
We have a lot cell mortality even before plating the cells and the ones that are surviving after plating are presenting vacuoles and are stressed. We continue to see debris and mortality.
We changed with new solutions all our media, we autoclaved our instruments, we changed many "lot" of FBS. We tested another incubator, changed our coating. We did also a mycoplasma test which is negative after 2 weeks of culture.
During summer we had some changes in our cell culture facility (temperature changes, pressure, ventilation filter changes) and we are 4 users with the same problem (knowing that before summer for all of us, all was working well).
We notice that our media change color really fast (after less than one week after opening)
We changed I guess all and we don't have ideas anymore of what could be the problem.
Any ideas ?
Thx a lot
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Hello,
Do you remove meninges during dissection? and, Also, do you centrifuge your cell suspension? if yes, what's the speed and time ?
Thank you.
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Hi all, I am looking glass cilinders that have an open flat bottom and top. Preferably with the inner diameter in the range of 2 cm.
It's similar to the ones found on multi-electrode arrays for neuronal cell culture.
Thank you in advance.
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Sourcing glass rings or cylinders for cell culture, especially with specific dimensions, can be a bit challenging. However, there are several options you can explore to find the right products:
1. Scientific Supply Companies
Several scientific supply companies offer specialized glassware for cell culture applications. Here are some options:
  1. Sigma-Aldrich (Merck):They provide a wide range of glassware and may have custom options available. Check their catalog or contact their customer service for specific requirements.
  2. Thermo Fisher Scientific:Known for their extensive range of laboratory equipment and supplies, they might offer glass cylinders or be able to point you to a suitable product.
  3. Corning:A leader in cell culture products, Corning might have glass rings or similar items in their catalog. Their customer service can assist with custom orders.
  4. VWR (Avantor):They supply various lab glassware and might have the glass rings you need. You can check their website or contact them for custom solutions.
2. Custom Glassware Manufacturers
If standard options are not available, you might need to look into custom glassware manufacturers who can make glass cylinders to your specifications:
  1. Chemglass Life Sciences:They offer custom glass fabrication services and can create glass cylinders with the dimensions you need.
  2. AdValue Technology:Specializes in custom glassware for scientific applications. They can manufacture glass cylinders to your specific dimensions.
  3. Technical Glass Products:They provide custom scientific glassware and might be able to produce the glass rings with the required specifications.
3. Specialty Retailers and Online Marketplaces
Sometimes, specialty retailers or online marketplaces like Amazon or eBay can have what you’re looking for, especially from suppliers that specialize in laboratory glassware.
Contacting Multi-Electrode Array (MEA) Manufacturers
Since you mentioned that the glass rings are similar to those used in multi-electrode arrays for neuronal cell culture, it might be helpful to contact manufacturers of MEA systems directly:
  1. Multi Channel Systems (MCS):They manufacture MEA systems and related accessories. Contacting them might lead to a direct source or a recommendation for compatible glass rings.
  2. Axion BioSystems:Another company specializing in MEA systems. They may provide or suggest where to get compatible glass cylinders.
Tips for Ordering
  • Specifications: When contacting suppliers, provide precise specifications, including inner diameter (2 cm), height, wall thickness, and material type.
  • Quantity: Mention the quantity you need, as custom orders might have minimum order requirements.
  • Usage: Explain the intended use (cell culture) to ensure the glass meets the necessary quality and sterility standards.
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Hi everyone,
I'm interested in finding a protocol for detaching neurons in culture for use in flow cytometry. The main issue I can foresee is in the production of a single cell suspension. Any help would be greatly appreciated.
Tom  
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This is quite insightful. I am also trying to process neurons for FACS. It would be great if @Thomas you can tell me which one worked well for you? And what was the protocol. Any help would be really appreciated.
Tripti
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I am planning on staining neuronal cells in culture to visualize neurites. Thermofisher's Vybrant-CM DiI seems the best fit for my requirements. However, I am unable to find any images or papers that show the use of the this stain on neuronal cells. I am now reconsidering the usage of this stain, and am also considering the Vybrant DiI solution or the DiI crystals also sold by Thermofisher. If you have used this with neurons, please comment on it!
NOTE: I aim to use a non-injectable stain.
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Kavya Kalpana Ganesh thanks for taking the time to answer! I'll let you know how it turns out for me.
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I am wondering if there are any commercially available fluorescent stains for live imaging of astrocytes derived from hiPSCs. We are co-culturing neurons and astrocytes and would like to stain the live cells to determine the change in their populations over time.
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May consider using Permai fluorescence dye.
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I would like to infect a high proportion of mouse cortical neuronal cultures from P0 pups with AAV for a biochemical study while avoiding toxicity. I would like some suggestions on the MOI (ratio of viral genome copies per cell) to use. I am using a AAV-DJ purified via iodixanol ultracentrifugation. I have cells plated in 6 well plates at a density of 10^6 cells/well and in 24 well plates at 150,000/well.
I know there are at least a handful of publications that have made comparisons of different MOIs (see below). The best would be to make my own empirical determination, but wanted to see if anyone had any input, as the some of the publications seem to suggest pretty large ranges in appropriate MOIs( ie 2*10^4 versus 1*10^7). Thanks!
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Dear Researcher,
Regarding your inquiry about the optimal multiplicity of infection (MOI) for infecting primary mouse cortical neuronal cultures at postnatal day 0 (P0) with adeno-associated virus (AAV) serotype DJ, it's essential to approach this with a nuanced understanding of both the viral vector characteristics and the specifics of your experimental system.
AAV serotype DJ is a synthetic serotype, known for its robust and broad transduction efficiency across various cell types, including neurons. This attribute makes it a preferred choice for gene delivery in neuroscience research. However, determining the precise MOI for primary neuronal cultures, especially as specific as cortical neurons from P0 mice, requires careful consideration of several factors:
  1. Viral Vector Preparation: The quality and titer of your AAV preparation significantly influence the effective MOI. Ensure your viral vector is of high purity and accurately quantified. A titer ranging from 10^12 to 10^13 genome copies per milliliter (GC/mL) is commonly used for neuronal transduction.
  2. Target Cell Sensitivity: Primary mouse cortical neurons, particularly from early postnatal stages like P0, can be more sensitive to viral infection compared to immortalized cell lines or neurons at later developmental stages. This sensitivity necessitates a cautious approach to MOI to avoid cytotoxicity while ensuring adequate transduction efficiency.
  3. Experimental Objectives: The desired outcome of your experiment, such as overexpression or knockdown of a specific gene, will also dictate the optimal MOI. Higher MOIs may be necessary for robust gene expression but be wary of potential toxicity or off-target effects.
  4. Literature and Precedents: Empirical evidence from similar studies can provide a useful benchmark. For primary neurons, MOIs ranging from 10^5 to 10^6 GC/cell are commonly reported. However, variations exist based on the AAV serotype, the target gene, and the specific experimental setup.
  5. Preliminary Experiments: Ultimately, determining the ideal MOI for your specific conditions may require conducting preliminary experiments. Start with a range of MOIs (e.g., 10^4 to 10^7 GC/cell) and assess both the transduction efficiency (via reporter gene expression or target gene knockdown) and cell viability. This stepwise approach allows you to fine-tune the MOI for optimal results in your system.
In conclusion, while a starting point of 10^5 to 10^6 genome copies per cell is a reasonable baseline for primary mouse cortical neurons at P0 using AAV-DJ, adjusting this based on the factors outlined above is crucial for the success of your experiments. Ensure rigorous optimization and validation steps are included in your experimental design to achieve reliable and reproducible outcomes.
I hope this detailed overview assists in guiding your research endeavors. Should you have any further questions or require additional insights, please do not hesitate to reach out.
Best regards,
l This protocol list might provide further insights to address this issue.
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Hi everyone
I am currently investigating the effect of iron deficiency on neuronal cells (SHYSY5Y to be exact). For this, I need to create an environment of iron-depletion in-vitro. While past publications have used iron chelators in media, this involves considerable quality control. This is why I am choosing to mimic an iron-deficient environment through serum starvation.
I am currently struggling to find publications outlining a validated method of serum starvation to achieve this, which would be of great help, as a trial- and error method in the lab is time consuming. Another concern is that other essential nutrients would also be depleted with serum starvation, that may affect any findings and therefore impact the validity of results. I am also interested in any iron-depleted media out there that I could potentially use?
Would greatly appreciate any advice, links to publications or methods I could follow.
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Ruchitha Venkatesh An effective method for preparing iron-deficient cell-culture medium without using chelators involves serum starvation, which mimics an iron-depleted environment. However, this approach may also deplete other essential nutrients, potentially impacting research findings. To address this, you could consider using commercially available iron-depleted media specifically designed for cell culture experiments, ensuring the controlled removal of iron while maintaining essential nutrient levels. Additionally, consulting relevant literature or protocols for serum starvation techniques in neuronal cell cultures may provide valuable insights and guidance for your experiments, reducing the laboratory's need for trial and error.
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I have isolated cortical neurons from mouse pups ( day1) and seeded on poly d lysine coated wells of 24 well plated (120000cells/well) and cultured in a medium composed of Neurobasal A, Glutamax, pen/strep and B27. After 2 DIV the cells appears like neurospheres ans they started to show processes and get connected to each other. why neurosphere like structures appear in primary cortical neuronal culture?
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I found the same issue in my neuron culture. How did you solve it finally, Divya?@Divya Vipin Menton
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We are plating primary hippocampal neurons from rats at post-natal day 0-2.  At plating 350,000 cells/mL with a total of 4 mls/plate.  We coat our plates with PDL and then with lignin.  At plating, the cells are placed in MEM + FBS + L-glutamine + Pen/Strep.  The day after plating they are switched to what we call neuron media containing Neurobasal A + B27 + Glutamax + FdUR + Uridine+ Pen/Strep.  We feed the plates every 3-4 days by removing 2 mls and replacing 2 mls of fresh neuron media.  The cells look great all up to the change right around 14 days.  After this change, like clockwork, the cells start dying the next day.  I make up the fresh media the day of changing and do not let it sit for more than 10 minutes in the waterbath.  Any suggestions why the cells do well for so long and then start dying at this last media change?  We are hoping to work with more mature neurons (day 14 or older) but so far have not been able to. 
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Hi,
I culture E18 rat cortical neurons. Switching to younger neurons may help. When I was troubleshooting my cultures I found that the plate coating made the biggest difference. We used to use PDL but we had inconsistent cultures and adherence issues.
We switched over to dPGA from Dendrotek (https://dendrotek.ca/products/centrifuge-tube) for coating our plates and our cultures were way better. Much more consistent adherence, cultures lasted longer and seemed to have more processes on the plate. We use it the same as PDL, 10 ug/ml in ddH2O at 4C overnight. I've gotten cultures to last for 10-11 weeks using this.
Hope this helps!
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Hi,
I am culturing primary Motoneuron from mouse spinal cord on Porn and laminin coated coverslips. By day 7 I get about 10% survival of the neurons. The seeding density was 5000 cells. What can I do to increase the survival of the Motoneurons.
please share your thoughts
thank you
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Hello Sonia, could you provide more information? Are you using embryonic or neonatal mouse/rat? Are you following a published protocol? White size of coverslips are you using or density of cells per mm2? What media with/without supplement? Just by the look of it it sure looks like the density of the culture is too low, but it's hard to say without the details.
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I am working on a protocol to measure calcium flux by synaptic signaling in live mouse neuron culture. I am using Fluo-4 AM for my stain, which I have seen others use, but it is not showing any flux or change in signal that I'd expect to see with movement of synaptic vesicles. Has anyone had success with this stain or suggestions for an alternative calcium stain I could use? Alternatively, any other ways to measure synaptic communication? Any help is greatly appreciated.
Thank you!
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Gabriella Saro The neurons look completely healthy and seem to be alive (I can see some movement in the cytoplasm during imaging). The staining is done in HBSS, and I have tried most of your suggestions in some form - but this makes me wonder if I have been staining them too early. I will try some later timepoints. Thank you!
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I have been trying to analyse neurons for spine density and size using ImageJ.The images are from fixed brain section taken using confocal microscope. but the background fluorescence is interfering with the analysis as the software is unable to distinguish between actual spine and background noise. What plugins/macros can be used to reduce noise and clearly outline the structure of the neuron. Please do not hesitate to ask for more details in case you think you could help me out ! Please find the iimage attached. Many Thanks, - Raj
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Try to use ImageJ, select "Process" > "Subtract Background" > "Light Background" + "Separate Colors" > Save the image. Hope it helps.
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Dear knowledgeable,
Have been doing neuronal cell cultures for many years now, (yikes). I've found it useful to "balance" my media in the incubator prior to applying it to the cells. This meaning approaching equilibrium of temperature (37dC) and CO2 between the atmosphere in the incubator and the media. Especially with Bicarbonate media this keeps pH in-check.
Have no exact sources but usually about 30minutes.
I recently started doing low oxygen cultures, where O2 is kept at around 5% (by flooding with nitrogen). I am not sure of the details of how much N2 is actually used to balance out the 02.
My question is therefore, how long time should I leave my my norm-ox (~20%) media in the incubator to reach low-ox (5%) equilibrium.
Thinking one of you clever chemists might be able to give an approximation?
Friendly regards, Staffan
Ps. For this example. Imaging a filled 50ml tube - Surface area ~5-7.5cm2, and the surface undisturbed ;)
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Bump!
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I differentiate N2a cells with 20 uM retinoic acid 1% serum in a 24 well plate. During the differentiation process that lasts 4 days, I change the medium every two days, but on the 4th day the cells start to die. How can i make cells live for a long time (10 days)?
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Chandra Somasundaram I am using Gibco DMEM low glucose. Actually I also tried to change it every day but the result did not change.
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I am trying to optimize the dose of drug for neuronal cell culture. How do I determine EC50 value? Suggest literature and easiest method and calculations. is checking the cell viability acceptable to determine EC50 values or have to consider other parameters also? . How can dose response curve will be used to calculate EC50? Please guide
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How do I determine EC50 value?
Use the drug dose-response curve to determine EC50 value. This curve is used to test drug efficacy invitro using cell lines.
Is checking the cell viability acceptable to determine EC50 values or have to consider other parameters also?
Checking cell viability is enough to determine the EC50 value.
How can dose response curve will be used to calculate EC50?
The EC50 is the concentration of the drug that provokes a response halfway between the baseline (Bottom) and maximum response (Top). It is impossible to define the EC50 until you first define the baseline and maximum response. You may refer to the link below to calculate the EC50 from dose response curve.
Suggest literature and easiest method and calculations.
You may also refer to the link below.
Best.
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Hello everyone! I am trying to stain mature neurons in culture for pre and post synaptic markers. I used to fix my cells 20 min at 37C with 4% PFA. Synaptophysin looks amazing unfortunately I couldn't achieve PSD95 puncta. I read that the best fixation strategy might be a short PFA fixation or an ice-cold methanol fixation. As I have several samples already fixed, does someone know if a post-fixation incubation with methanol could help me visualize the PSD 95 puncta? or does someone have the ultimate protocol? also with the antibody cat number?
Best
Telma
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Hi Telma,
I have the same problem, can you let me know if post-fixation incubation with methanol helps visualize the PSD 95 puncta?
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I am curently working on primay neuron cell culture and i read that D-arabinofuranoside inhibits the glial cells. Any other compounds that can inhibit the division of glial cells?
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You could use 5-fluoro-2’-deoxyuridine (FUdR) as a possible substitute for D-arabinofuranoside. Please refer to the research article attached below.
Best.
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Hi everyone,
I'm looking for RNA extraction kit for isolation from cultured cells. I'm working with neuronal cell culture (SH-SY5Y).
Thank you in advance for your suggestions.
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Dear John Hardy Lockhart,
Appreciate for your suggestion.
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Hi,
I'm doing neuronal cell culture (SH-SY5Y) and can not use antibiotics and antimycotics in my culture medium as it will affect to my following procedures.
I've tried to use sterile labware and materials, however, I still see contamination, specially, fungal contamination in both flasks and plates that the color of the medium becomes cloudy, and cells started to detach from the surface of the flasks and plates.
Can anyone suggest how to prevent and reduce this contamination?
Thank you
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You have to pay attention on each and everything you use, wear cap, mask, change gloves more often, solution must be sterile filtres. Just common sense care. Be more alert on cleanliness and sterility. Clean incubator more often, use autoclaved water in waterbath and in incubator, clean handles of every equipment you are using.
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I was wondering whether NAD+ (not nicotinamide riboside or nicotinamide mononucleotide) can enter cells when supplementing the medium of a neuronculture with NAD+
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Hello Fa Ro
You may please refer to the research article attached below. It will be helpful.
Best.
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Good day to all,
We are extracting and cultivating cortical neurons from P0-P2 rat pups. But they seem to stay depolarized in culture and we don't understand why. Morhologically they look good, healthy but they do not show spontaneous activity in MEA measurement or under patch clamp.
Could anybody give a hint what we are doing wrong?
for reference:
- P0-P2 rats, cryoanesthesia and decapitation, cortical dissection, cut in tissue chopper (100µm x 100 µm)
- digestion in accutase (20 min at 37°C)
- seed after membrane filtering (45-75 µm)
- seeding medium: DMEM with 10% FBS + Pen/Strep (only 3 hours for attachment)
- feeding medium: Neurobasal A with B-27, Glutamax (1 mM), Normocin
- medium change: 2x a week, only half is changed, no aspiration
--> Patch at 10 days: all depolarized (~ -30 mV)
--> Patch at 16 days: all depolarized (~ -30mV)
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Have you or somebody else has done the cortical neuronal culture earlier?
If No, then humorous factors could be one of the probable reason that led to depolarization of neurons. Since our culture media consist of minimal compounds that we have established earlier, In contrary to that our circulatory system carries thousands of the factors with varying level their synchronization affect the development of in vivo system.
if you think the same reason, I suggest to use the fetal/pups serum of rat that may help you to get good results.
Regards
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Dear scientists,
I have a question regarding self-renewal assay.
Instead of manually counting neurospheres in each well after seeding at clonal density
is it possible that counting is done automatically (for example with FACS)?
Have anybody tried it?
Thanks a lot for your answers and help.
Regards, Snjezana
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Also do keep in mind, I guess depending on how often you do these counts, that there are more automated options as well. The white paper below is for mammospheres but should translate to neutrospheres as well.
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I'm learning to culture primary hippocampal neuron from E17 mice. I use commercial coverslip from Neuvitro with pre-coated PDL and laminin. The soma of the neuron tend to aggregate together with entangled neurites. At DIV17, the percentage of mature spine is relatively low. My transfection efficiency with lipofectamine is also not high and cause a lot of cell death.
Can anyone give me some suggestions? Or recommend some protocols and troubleshooting tips?
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Hey
If you would share your protocol I will see through it. Why do you use Lipo2000?
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I see that many papers include analyzing calcium in their methods when the differentiation of iPSCs to neurons is taking place. Why is this necessary?
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Basal calcium is usually low in the cytoplasm of unstimulated cells, most of it is stored in the ER and in the extracellular environment. A good way to assess the differentiation of iPSCs to neurons is by testing their ability to respond to external stimuli or to fire spontaneously after synapse formation in culture. These activities would determine calcium transients that can be measured and quantified - e.g. comparing the response to the baseline (stimulus vs absence of stimulus). You can check this paper about an iPSCs-derived disease model.
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I see small aggregation-like particles in primary neuron culture from the hippocampi of rats. Can anyone suggest what are these ? And can we go with drug or other treatment assays with these particles in culture and they will not affect the experiment?
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Aggregates of dead cells, most likely due to excessive trituration or too much time spent in dissociation medium. I don't think coating is an issue (there are many attached neurons on these pictures).
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Hello,
I will develop materials for the repair of nerve damages in my master thesis, and I am planning to use neural cells for cell culture experiments. I have cell culture knowledge, but I have never worked with any kind of neural cells. If you have experience on this subject, can you tell me which cell line would be more proper and easy to handle for me and what should be considered differently from basic cell culture in this process?
Thank you!
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I have no experience with neural cell cultures. Moreover, it will be extra difficult for me to obtain and purify a primary culture. I think it is better to prefer to buy cells commercially.
Thanks a lot for your answer!
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Hello all,
I am currently trying to culture cortical neurons in media free of T3 in hopes to observe change when supplementing the cells with different concentrations of T3. I have looked up some literature and all I can seem to find is use of serum-free DMEM. Has anyone tried this before and can give me any insight on cell health? Or just any links to medias that can support neuron growth but also lack serum/T3?
Thanks in advance :)
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If you require T3-free media, perhaps you can formulate a custom serum-free supplement that omits it from the mixture. If you are going to use a lot of it, please reach out to our company, and we might be able to create a batch of our SM1 (our serum replacement supplement for neurons) that has T3 removed. Thanks.
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I am new with this technique. It seems that different cells are responding to my drug with a very different ratio kinetics in my calcium imaging experiment. do they mean anything?
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I’m not sure what you mean by “ratio kinetics”, so I can’t give a definite answer. To me, kinetics would refer to the speed of change in fluorescence. For that, actually, fura-2 isn’t a great dye, because it is intrinsically slow. Check out papers by Wade Regehr from the 90’s on this. If you want to study kinetics, you need fast, low-affinity dyes, like fluo-4, and you need a fast detector too, such as a photodiode or photomultiplier tube. Most cameras are too slow. It will be hard to detect a change in Ca channel kinetics even with the best system. You’ll want some solid positive controls (e.g. applying EGTA to accelerate the kinetics).
Ratio also may mean different things, especially with fura-2. If you are calculating the ratio of fluorescence at 380 nm emission compared to 340 or 360, that is telling you about the absolute concentration of Ca. You should read Grynkiewicz and Tsien about how to translate the 380/340 ratio to concentration. Additional measurements are needed. The bottom line would be, if the ratio changes with your drug, then Ca concentration is changing.
Another ratio associated with fura-2 is when you stimulate twice, and compare the amplitudes of the two fluorescence peaks. Because fura-2 has a high affinity, a ratio close to 1 would indicate the Ca concentration is quite low, in the low 10’s of nM. If the ratio is low (i.e. the dye is saturated), then the concentration of Ca is high, in the 100’s of nM to uM.
If you can give more details about your prep and measurement system, I might be able to be more specific.
-Matthew
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I am trying to grow motor neurons on coverslips and chamber slides, and every time I pipette out the spent media and pipette in new media with a P1000 pipette, a lot of neurons seem to detach despite careful pipetting techniques.
Is there another way I can change media (instead of using P1000 pipette) without risking so much neuronal detachment from the coverslips?
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Seung Woo Baek This problem is pretty common. You need to be very careful when pouring or adding up the fresh media. While removing the used up media, by default we do tilt the flask so that media could collect in the corner and that does not cause any problem. But cells detachment happens because of the force of fresh media which is being poured directly over the cells surface. So you shall add media gently over the wall of the flask interior, while having the flask tilted.
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I have been trying to depolarize the mouse hypothalamic cell line, GT1-7 in order to measure GnRH release in media. However, any efforts to depolarize the cells does not produce any increase in GnRH above baseline.
I have tried exposing the cells to a 55 mM K+ solution (osmotically balanced) for 15 min, and I have tried using Kisspeptin at concentrations of 1, 10, 100, and 1000 nM (for 1 hr). Both methods have been used in the literature to cause GnRH release. I treat the cells in 200 uL to concentrate any GnRH release as much as possible and measure around 30-40 pg/mL at baseline. I culture the cells on a collagen coated 24-well plate in DMEM+10% charcoal-stripped FBS overnight before treatment.
I'm using a GnRH EIA Kit from Phoenix Pharmaceuticals that has been used in the literature to measure GnRH (https://www.phoenixpeptide.com/products/view/Assay-Kits/EK-040-02CE).
I'd be grateful for any tips of hints on how to depolarize these cells or any insight into what I may be doing wrong.
Thanks!
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I send you two papers about kisspeptin and compared them to GnRH and hCG.
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Hi everyone,
so, we always had good working primary cortical neurons preps (embryonic) and no issue with culturing them to DIV10-14. However, the animal facility now moved to a different location and instead of being next door it's now a 10min cycle ride away. Interestingly the issue of sudden neuronal cell death at around DIV10 started around the same time. Has anyone experienced something similar before?
We also started adding AraC on DIV4 at 2uM (final conc.) to the cells as we experienced overgrowth of non-neuronal cells but according to the literature this is a well accepted concentration by the neurons. I leave it on until the next media change which happens 3days later.
I use the new B-27™ Plus Neuronal Culture System from ThermoFisher with 50% media change every 4days. Plating (4-5h) is done in DMEM, 10% FBS, P/S.
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I agree with Edda in NOT dissociating tissue that needs to be transported. If you can do dissection in your local building it will greatly help with survival.
Lot-testing your neural supplements is always something to check. NeuroCult SM1 from STEMCELL technologies undergoes testing to ensure lot-to-lot consistency in performance.
Thanks
Jason
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Hello,
I am trying to induce the over-expression of alpha synuclein in mouse neuron cultures. I want to try to see whether a specific AAV or Lentivirus would help me induce over expression. Has anyone ever done this before?
If possible, it would be very helpful to know the capsid/serotype, best promoter, time to see expression, duration of expression and optimal infection efficiency.
Feel free to share all sorts of experiences/ protocols.
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Dear Ebony,
I have tried AAV and lentiviral expression of alpha-synuclein in cultured neurons and both work well. If you want to infect all the neurons in the culture, I would use AAVs (e.g. serotype PHP.B). However, if you want to control the number of infected neurons I would recommend lentivirus, because you can adjust the titer more easily. For lentivirus expression I have used the classical FUGW backbone (ubiquitin promoter).
Hope this helps,
best wishes,
Chris
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Hi!
I have been trying to inhibit NPC proliferation in human iPSC-derived neuronal cultures with compounds such as Ara-C, FUDR and DAPT. All the compounds kill a considerable portion of the cells. Lowering the concentration will kill less cells and allow more proliferation. Is this how these compounds are supposed to work? Is there any way to just promote neuronal maturation without killing the cells?
Best, Noora
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Dear Alexandr,
Thanks for the answer! During neuronal maturation, I have added BDNF, GDNF, and CNTF in the medium.
Best, Noora
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Hi all, I'm currently trying to isolate oligodendrocytes from GA62 (term is GA70) guinea pigs and am having a bit of trouble getting them to survive. I take the tissue and digest with papain and then plate at 10million/T75 flask in DMEM with anti-anti and 10% FCS. This step is fine and they reach confluency around DIV 10-12. I preshake them for an hour to remove microglia and then shake overnight, and then plate in a petri dish for an hour in the incubator to remove any residual glia. I plate them at 20,000 cells/well in a ornithine coated 24 well plate in a mixture of DMEM/apo transferrin/insulin/sodium selenite/D-Biotin/hydrocortisone and 20ng/mL of PDGF-AA and bFGF. I've added 10uM/well Ara-C on day 2 after plating and did a complete change to remove it 3 days later.
I then remove the growth factors and replace with T3.
I've attached some photos from DIV 6 after plating and before growth factor removal but they don't look great. Can anyone offer any suggestions? I was wondering about adding NT-3 into the mix, or maybe not leave the Ara-C in for so long? Any help would be appreciated :)
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One very basic tip depends on the % CO2 you are using. Without serum, DMEM has a bicarbonate concentration that relies on 10% CO2. If you want to remain at 5% CO2 go to a DMEM/F12, 50/50 medium, which has the appropriate bicarbonate for 5% CO2. Past that, I have not worked with microglia, but trying less time in Ara-C is something to try. Good luck.
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#laminin #poly-D-lysine #coating #eplate #neuroncellculture #RTCA
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Kindly, read this interesting paper, hope it will be useful.
Regards,
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I have been trying to troubleshoot two issues,1) low yield of neurons 2) avoiding cell death after day 10 of culture.
I'm trying to isolate mouse cortical neuron culture in a new lab from P(0-1) pups onto PLL coated coverslips. I use papain digestion (30min) at 37deg. Add FBS and rinse the cells to remove FBS and triturate in HBSS. I use 1% bsa to layer the digested/triturated tissue. Could you provide any suggestions/or share any working protocol. I have been searching several forums for a response and I've been trying several alterations, it's been 6 months.
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Thank you Misha. I did try this protocol this time. I somehow found the neurons to cluster. I had this issue in the beginning stages. I changed from PLL to PDL and used a fresh dilution of PDL (50µg/mL). They seem to be fine on the dish but don't like the coverslips. I used HCL instead of nitric acid, baked at 50deg for 3 hours before rinsing with tap water and thrice with ddH2O. I coated with PDL for 1hr at RT before washing thrice with ddH2O and air drying. I find more death on the coverslips, even if I seed 600K in 35mm dishes.
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Hello everybody
In our iPSC-derived neuronal culture we use Maturationmedium, supplemented with B27+.
As Thermo Fisher run out of stock and can deliver earliest in May, I wanted to ask whether you have alternatives for this supplement.
We already thought about normal B27, or the one from R&D (https://www.rndsystems.com/products/n21-max-media-supplement-50x-_ar008).
But in both of them the "+" seems to be missing.
Do you have any other ideas, or do you know which ingredients are needed to make B27+ out of B27?
Best wishes
Katharina
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The B27+ can easily be replaced with B27 although this MAY result in decreased survival of the neurons (Personally, I have not seen a huge difference in neuronal survival between B27 and B27+). A better option is using MACS Neurobrew from Miltenyi. The composition is very similar to B27, in fact, this is a better alternative to B27. My understanding is that the MACS Neurobrew is comparable to B27+. They have a formulation without Vitamin A as well if needed.
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Hi everyone,
I have been trying to get a gigaseal from primary cell-culture cells from rat’s DRGs, but without success. I don’t know exactly what the problem is.. I have tried after 2 hours form getting the cells, after 24 hours, and 48 hours, I have changed the glass used to fabricate the pipettes, (I tried both thin walled glass and thick walled glass), I have tried with different pipettes resistances (from 2MΩ to 10 MΩ) but without success.
A PDF that I prepared is associated to explain what I have done, containing screenshots of what I get in the “SutterPatch” Program, and picture of the pipettes I used.
Do you have any recommendations or a solution to help forming gigaseal ?
Thank you in advance!
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Thanks very much, we will try this and see what we will get
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Hi there, I'm isolating neurons from PND1 guinea pigs and have noticed a bit of debris in the early days (this is day 4 plated) and a lot of variability between plates. Current protocol is dissociate with 50uL Trypsin in 5mL HBSS (wondering if I should add DNase?), spin and wash pellet and plate at 60x10^4 cells/well in a 24 well plate in Neurobasal/B27/L-glut/PenStrp/HEPES/10% horse serum. Leave them overnight then change to a serum free media. I've attached a photo, any advice is appreciated
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This is clumps of cells...
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Hello, I come late in the conversation, but maybe it could still help. I work with LUHMES for ~2 years now and with the following tricks I do not have many problems:
For coating, I use the concentrations recommended by ATCC, but I do either the 2 steps procedure (PLO in ddH2O overnight at RT, rinse, 3h human fibronectin at 37°C, wash and dry) or the 1 step procedure (PLO + fibronectine in ddH2O, overnight at 37°C, wash and dry). Both work the same. Coated material is store up to 1 month at +4°C inside sterile bags.
For recovery from frozen stock, I seed 2Mio cells in a T75. the 1st 2 days they look rare but then they recover very well. Medium is changed every 2-3 days, completely.
Yes cells die if they grow more than 80-90%. I split often 1/5 to 1/10. They grow slowly the first days but then very well.
For detachment I prepare the 0.05 trypsin-EDTA as described in the datasheet, which is diluted 1/2 with PBS prior to use. I rinse the cells with medium without bFGF, let 3min at 37°C with 5ml of diluted Trypsin-EDTA. Then very important I do not tape the flask. I pipette the trypsin solution on the cell layer to detach them, transfer in a tube, rinse the flask with medium without bFGF and pool with cells. Then centrifugation 7-8min at 130g (or 5min at 1000rpm). Important, I remove almost all medium but not all, then I flick the tube to resuspend the cell pellet as much as possible. Then only I add 1ml of medium to finish resuspension. Otherwise cells clump and it is almost impossible to have an homogenous suspension.
Best
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Currently, I have a problem to isolate total RNA from mouse primary cortical neuron culture. I am seeding neurons to poly-L-lysine coated coverslips and seeding 500k cells per coverslips in a 6-well plate. For RNA isolation, I was scaping 3 coverslips under 1 ml trizol. After that, I was proceeding with Direct-zol RNA isolation kit protocol. But there was no RNA or sometimes very little RNAs such as 20ng/ul after isolations at many times. Cells were looking confluent and healthy before scraping. Therefore, I am not sure about which stage the problem occurs. I would be so grateful to get an extra idea/tip about this. Is there anyone doing RNA isolations from primary neuron culture and managed to solve this kind of problem?
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Hi Ayca,
I agree with Suraiya that in theory for your purpose it would be better to grow the cells directly in the well plate e.g. as a mixed culture containing glia and neurons. We had problems once with scraping cells from coverslips. However, still I think that with your feeder-approach having the neurons on coverslips you should be able to extract more then enough RNA. I was currently using a mixed culture of 800 k cells per 6 Well. Cells were harvested at DIV9 or DIV21 using QIAGEN lysis buffer and RNA extraction was perfomed with QIAGEN RNeasy Kit. In the past, I made best experiences with the RNeasy kit. I also combined it with TRIZOL lysis and extraction and it worked very well. I don't have experiences with the kit that you have mentioned. However, with the RNeasy kit I usually get 3-5 µg total RNA of high quality (RIN>8) from these 800 k cells.
Maybe you could try this.
Best
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I performed primary neuronal cell culture successfully in 25-T flask, but when I turned to do it in 96-well plate, I faced this problem with my cells, I changed their media (neurobasal, b27, glutamine, FBS and pen/strept) on day 4 with 50% exchange of the old media for the fresh one, however it caused cell death on day 7 like what you can see in the pictures attached here, i guess it could be because of the plate coating. I had to do it differently from how i used to do with my flask.. I use poly-l-lysine and its time of expoture is around some minutes. do you also think that it's the right reason?
I also newly changed my trypsin source which it's stronger than the previous one and I can see more dead cells during counting, may it shorten the living cells' life span?
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I would suggest longer time if coating. But also removing glutamine after cells settled properly (like 3 days in vitro). Immature neurons are not sens, but as the cells get matured the glutamine get cytotoxic. Good luck!
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Hi,
I am currently running an experiment that involves a 10-minute incubation of live cultured neurons in PBS containing magnesium and calcium on ice. Following immunocytochemistry for the neuronal marker MAP2, they appear either 'streaky' and inconsistent or blebby throughout the dish of cells.
The PBS-MC recipe I use is:
0.137 M NaCl
2.7 mM KCl
4.3 mM Na2HPO4.7H20
1.4 mM KH2PO4
1.33 mM MgCl2
0.133 mM CaCl2
I have tried adjusting the magnesium and calcium concentrations, have tested the osmolality as well as the addition of sucrose. I have also tested whether the ice is too cold, however, even room temperature gave the same results. I have also tried without magnesium and calcium. In separate experiments, I used aCSF with an osmolality of 290 mmol/kg, adjusting the PBS-MC recipe to be close to this value had no difference.
Any suggestions would be great
Thanks!
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Dear Courteney!
Please You try following potocol:
1. Dissociated Hippocampal Neuron Culture
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I am trying to characterise GABA developmental switch in iPSC derived neurons using Ca2+ imaging. However, as I add GABA agonist, I observe the spontaneous activity of several neurons being abolished, and addition of gabazine in those neurons brings up the transients again.
I am only able to find fewer neurons in culture that show Ca2+ elevation upon GABA addition.
Any idea why this could be happening?
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Addition of gabazine removes this inhibition and allows the excitatory network activity to return.
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Hello,
I've been trying to isolate RNA from hiPSC derived neuron culture (70 days old) with GenElute™Mammalian Total RNA Miniprep Kit but I could not obtain a good yield even though I had a good amount of neurons in the culture. I isolated from a single well of 6-well plate which was not fully confluent yet the networks were extensive and dense. I am sure of my homogenization step since there were not any particles.
Did you experience something unusual while isolating RNA from dense neuron cultures and do you know a way to recover RNA from the column if the sample clogged it?
Thanks in advance
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I see, thank you:))
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Hi !
I'm working on a protocol utilizing mDC from MUTZ-3, I didn't find information about time of survival for these cells after the protocol of différenciation. In want to prime these cells with PMA or LPS and put them on a neurons culture, I also didn't know how much cells I should put in it.
Thanks for help
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Hello, Tristan!
MUTZ-3-DCs displayed a similar expression profile to MoDCs (Fig. 2), as MUTZ-3 lost expression of CD14 during the 7-day culture period and acquired CD1a expression.
Figure 2
Changes in CD14 and CD1a expression during differentiation and maturation of monocyte-derived dendritic cells (MoDCs) and MUTZ-3, as assessed by flow cytometry. CD14+ peripheral blood monocytes (a) were differentiated for 7 days into immature MoDCs (b) and subsequently stimulated with a proinflammatory cocktail for 48 hr (c).In a similar fashion, MUTZ-3 cells (d) were allowed to differentiate into an immature DC-like phenotype (e) and were further stimulated with a proinflammatory cocktail for 48 hr (f).
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I am looking for a b-secretase inhibitor that was used in hiPSC's neuronal culture.
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BOC Sciences provides a wide range of research chemical and biochemical substances, including inhibitors, GMP products, impurities, and metabolites. Our beta-secretase inhibitors products include MK-8931(also known as Verubecestat), LY2811376, LY2886721.
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Hi all,
I just started to prep mouse cortical neutrons for E16.5 and in my second prep I had a massive fungal contamination. Since these preps are quite precious, I wonder if I could add an antimicotic to my supplemented Neurobasal medium to preven contaminations in my next cultures.
Any personal experience or suggestion will be much appreciated.
All the best,
Ernest
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Puromycin may help, its been shown to inhibit yeast growth. It has not negatively affected our cultures.
Personally, I decreased contamination in my cultures by improving aseptic technique and treating the incubator water bath with Copper 10g and EDTA 0.2g in 10L H2O. That cut way down on the frequency of culture contamination.
We also grow primaries in covered image chambers stored in poly trays with lids. Both of those are UV'd for at least an hour before getting any substrate.
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would like to know how to differentiate them and how are the phenotypic changes? do they give rise to dopaminergic or glutamanergic phenotypes?
what would be the agents to induce differentiation in these cells?
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Hi Abu Bakkar Siddik, I would try differentiation for 48hrs up to 96 hrs even. Also good differentiation markers include Synaptophysin, NeuN, and PSD95
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We are interested in sorting interneurons from human brain (cortex). I´m thinking about the possibility to use GAD 65&67. Does anyone have any comments? 
Thank you!!
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Would like to share my latest publication with neuro scientists- The issue of energy teleportation within organs parts is demonstrated- i.e.: Hair follicle teleporting energy to an isolated cut hair shaft. Comments are appreciated:
To download link to PDF or URL icons under journal picture- Thank You.
EVIDENCE OF TELEPORTED BIOELECTROMAGNETIC ENERGY TRANSFER IN A HUMAN MINIORGAN CAUSING DELAY IN CRYSTALLIZATION  Abrahám A. Embí BS *1  *1 MBA, 13442 SW 102 Lane, Miami Florida, USA 33186  DOI: https://doi.org/10.29121/granthaalayah.v8.i6.2020.484
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Good day, I am using human neuronal culture differentiated from hiPSC's. I want to freeze them for future usage. So far I had trouble with keeping the cells alive throughout freezing and unfreezing procedure. Can anyone recommend a good way for doing that?
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we do this routinely, you can find protocol in the methods part of this paper.
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I have been trying to get Ciliobrevin D to inhibit dynein motor activity in primary neuronal (cortical and dorsal root ganglion) cultures. I am tracking axonal transport of lysosome as a proxy for dynein activity (Ref: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436090/). I dissolved Cil D in DMSO at 50 mM and stored stocks aliquotted at - 20 less than 2 months ago. I used it at a final concentration of 25 uM or 50 uM and treat cells for ~ 45 min before tracking lysosome transport. But there is no inhibition of transport in both cortical and DRG cultures. The imaging was done at 37C with 5% CO2. I also tried Ciliobrevin A at 60 uM under the same conditions but had no luck with that either. Please share if you have experienced this problem before or have any suggestions or a different drug that has worked for you. Thanks!
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I found this description in a paper ( ).
"We observed that following reconstitution, after 1 week at 4° or 3 months at −20°, the drug lost efficacy based on its effects on axon extension and growth cone morphology. Thus, it is recommended that freshly reconstituted Ciliobrevin D be used for experimentation."
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Does anyone have experience with the Olympus cellVivo weather chamber for monitoring neurite growth over hours/days?
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sorry not
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My cells are cultured primary neuron from fetal rat, transfected with GFP-HA-double tag surface protein at DIV 7.
IF was preformed at DIV10-12, fixation with 4% PFA 15min and block with 10% Donkey serum. 594 fluorenes marked HA tag.
below is my picture captured with 60X microscope. The first picture is kind of OK but most of the rest is quite ugly. I am new to this and want to know what happend, would you please help me figure out?
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You have to try with more dilution in secondary antibody
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For a better understanding of our experiments in cholesterol efflux and glucose captation, we would like to reproduce them on neurons, but we have no experience on them, that is why we would like to start with the easiest neurons to grow.
Thnak you!
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Hi Olga,
there are neuronal(-like) cell lines available, such as SH-SY5Y. However, you would have to check if your transporters etc. are expressed. Alternatively, you could use cortical neurons derived from embryonic or neonatal mouse brains. This needs some practice and for sure you need a lot of material for that (as well as mice).
Good luck!
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Hello,
I would like to mark the type of cells which are dead and alive after a LDA on neurons culture. How could i do ? Is it possible to do an immunofluorescence on cells after a LDA?
Thanks for helping !
Louise
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Calcein is exited at 488, propidium iodide is exited with a peak at 545, you still have DAPI 350 and Cy5 647 excitation windows to stain nuclei and protein of interest. As both calcein and PI staining remain florescent after fixation you can either ignore 488/545 channels and focus on DAPI/Cy5 or incorporate them into your experiment to distinguish the signals of your interest in living and dead cells. So it only depends on your microscope or facility core to have Cy5 excitation laser/light cube.
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I have been doing cell culture with B50 cell lines recently and the cells have not been showing the expected neuronal morphology, even after reducing the serum concentration. I just happened to do a check of the osmolarity of my cell culture solution and turned out to be 407 mOsmol/kg, which seems to be quite high compared to literature values.
My DMEM contains: 2.5%FBS, 2mM Glutamine, 10uPenstrep, Na-bicarbonate (3.7g/L), Sodium Pyruvate and HEPES.
I am adding too many things in the plain DMEM? Does anyone have an idea of what adverse effects this high osmolarity might have on neuronal cells? Could this be the reason for the bad cell morphology?
Will be thankful to any insights on this!!
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About 20 years ago, we found, but not published, that about 10% increase in osmolality leads to activation of "volume-activated chloride channels" in HeLa cells.
Thus, 330 mOsM/kg and higher osmolality will affect ionic homeostasis, various ion (primary Ca2+)-dependent pathways, and cell development.
To reduce your media osmolality by 100 mOsM/kg you can either dilute your media with H2O (by adding about 100/407*100% = 24% of additional volume with water), or to reduce concentrations of your initial components (e.g., by decreasing concentration of Sodium Pyruvate by about 50 mM).
What you choose depends on what component concentrations are more important for you in the media.
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We need to buy new CO2 incubators (specifically for work on bone marrow-derived DC and macs) and ESCO reps offer us these incubators that are substantially cheaper than similar models from better known suppliers (e.g. Thermo). No one here in Uruguay has experience with ESCO incubators, hence the question. Specific points: are these incubators OK in terms of avoidance of microbial contamination (is their 90ºC self-sterilisation cycle good)? Are they stable with respect to mechanical vibration (which activate dendritic cells)? Is their temperature control good (in spite of being air-jacketed)?
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In general esco co2 incubators working but other brands viz Thermo is better. Now we are getting the issue with sensor viz. Humidity sensor and CO2 sensor (which IR sensor) level in incubator. life of sensors seems to be maximum 2 years.
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Hey,
We are looking to study amyloid beta (1-42) and prion protein in a co-culture and separate culture system on neurons and astrocytes.
There seem to be a lot of different amyloid beta preps out there, I was wondering if anyone had an opinion or has had success with a certain companies preparation.
Thanks
Igal
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You can have a look at one of our recent paper for the preparation of amyloid stock and let me know for any concern.
Good Luck!!
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What is the best possible method to knockout genes fro a primary DRG neuronal culture? ..Apart from extracting DRGs from a knockout mouse..Since a knockout mouse is unavailable for the gene that I am studying..
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Hi Divija,
For CRISPR to "work" cells do not need to divide. The problem with CRISPR in primary neuronal culture is the relatively low efficiency of transfection plus the uncertainty of having a partial or complete disruption of the gene (only one or both alleles affected). That's why knockdown the gen transcript by shRNA, RNAi or miRNA is the most common choice.
Cheers,
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After plating the neurons look okay but they all migrate and clump at the edge of the plate. How can I keep them at the center of the well? I am using the 6-well plate. I was told to place a drop of 0.5 ml of resuspended cells at the center, but last time I did so the drop got dispersed when I was moving the plate. I am worried that there may not be enough medium to cover all neurons.
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I have struggled with the MDA assay for a while now. My samples are SHSY-5Y neuronal cell lines and i treat them with 500 uM hydrogen peroxide, 1.5 mM potassium bromate and 500 mM Ethanol to induce oxidative stress. I have tried the 2 methods attached, the first being a sigma assay kit yet the standards seem to be fine as there is the development of the pink colour and a nive standard curve afterwards but no reaction so far have been noticed in the treated samples. Any suggestions from previous experiences? or any proven alternative method?
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I also have a same problem with plasma and serum samples. Some of the samples react to produce color while others failed to develop color. I could not understand why it happens.
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Hi everyone,
I am trying to see GTP-tubulin inside my neurons cultures (from adult mouse DRG) by immunofluorescence, but I have a problem: a lot of neurons detach from coverslip glass and, when i look at them under confocal microscope, I can find only few and in bad state neurons. The remaining neurons are well stained, so primary and secondary antibodies work well.
Briefly the protocol:
1. Collection of DRG, disgregation, isolation and seeding of neurons is not a problem, I follow a protocol that, with normal immunofluorescence is good.
2. Neurons are seeded on coverslip coated with poly-d-lysine (I put a 100µl drop of poly-D-lysine on the coverslip for 15minutes, wash 3 times with water, completely dry the coversleep, seed 100-150 drop of neurons suspension). I think that this could be a critical point: the substrate.
3. The protocol for GTP imunofluorescence is described here: https://pdfs.semanticscholar.org/7fe6/5314198fdc2ac927ed269d760859d2dd35b3.pdf :
- Permeabilization for 3minutes in PEM-G + trition x100 0.05%
- Wash twice with PEM-G
- Add primary antibody MB11 (in PEM-G + 2g/L BSA)
- Wash 3 times with PEM-G
- Add secondary antibody (in PEM-G + 2g/L BSA)
- Wash 3 times with PEM-G
I can see quite all my neurons detaching from coverslip during this last washes after secondary antibody.
How can I improve the attachment of neurons to the coverslip?
Thank you very much
Alessio
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Hi Alessio,
I guess you are following the Dimitrov's 2008 protocol, which was performed in cell lines. I have never performed it in primary neurons, and I feel this staining protocol is extremely harsh. However, it has been reported to work inn neurons by Hirokawa's lab:
You will need to start the procedure with as many healthy neurons as possible. Therefore I would do the PDL coating on the whole coverlip overnight at room temperature and next morning do the washing, drying and finally add the laminin over the whole cover for a couple of hours at 37 C. Could you concentrate the 1ry and 2ry antibodies and reduce the incubation time? Any chance of conjugating the MB11 antibody to a fluorophore to avoid the second incubation? Perhaps this is already commercial.
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Dear all,
I patch dissociated neuronal cultures and would like to have more precise overview of neuronal types. I can distinguish neurons while patching them but it would be more helpful to know approximately which type I am going to patch while I am looking for one.
Do you know any staining (not harmful) to differentiate types and not to interfere with the physiological properties of neurons?
Or at least to tell if it is excitatory or inhibitory neuron?
Thank you a lot in advance!
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You can distinguish interneurons by the morphology. You may want to try staining fixed neurons with CTIP2 for CA1 and DG neurons once and see if you can find any morphological signatures usable for patching. I think there is a marker for CA3 neurons but cannot remember at the moment.
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I've been on neuronal cell culture to study neuroinflammation, but there has been a debate on neuronal cells not being relevance or giving significant role in neuroinflammation. It should have been immune cells like microglial cells.
But what if I want to proceed with neuronal cells, what are the markers that can be used other than inflammatory cytokine and chemokine?
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Expression of TLR4 in neurons is mostly in non-detectable range (see ). So if you are treating pure neuronal cultures with LPS, you would not see a significant alteration in cyto/chemokine profile. However, in many cases, primary neuronal cultures are contaminated with astrocytes, which does respond to LPS stimulation. A better model would be to co-culture neurons with mixed glia.
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I wish to look at gliosis in a dish, basically the neurons start becoming apoptotic and glial cells come to the rescue. But if I have a patient neurons, how do I mimic this event? Any idea what can be used a control?
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