Neuron Culture - Science method

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)

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

Dear All,

In most protocols who maturate neurons from IPSC-derivered NPCs (neuronal progenitor cells); they use NEAA in stemcell stage and NPCs induction phase, some of them use NEAA in NPC medium as well. However only a limited number of them using NEAA during neuronal maturation from NPCs.

Is there a spesific reason that we dont use NEAA during neuronal differentiation or is it just because initial paper was like that and people followed it?

Because it seems like NEAA are harmless and support cellular growth in all cell types.

thank you for answers

Yagiz

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?

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

I want to test antioxidant activity of a compound in neuronal cell culture and compare it with vitamin E (or Trolox) and vitamin C effects. Which concentrations of each would your recommend? Or which publications would you recommend?

Thanks in advance!

As soon as seeded (from neurosphere phase) on PEI (0,1%)/laminin (20 mg/ml)-coated p24 well plates, the MN precursors were struggling in both surviving and differentiating into healthy mature MNs. We need to point out that after having dissociated the neurospheres the live count with trypan blue was low, around 50%. We tried to investigate the nature of these crystal-like formations that were arising after two days in culture, in these ways:

- by moving some media from the NPC well in a new p24 well plate

- by coating again with PEI/laminin and placing new basal media or new differentiation media

We got no results. We then started thinking that these crystal-like structures were not a consequence of contaminations or supplement precipitation. Instead we assume that they are cytologically-related, for instance uric acid release of apoptotic cells eventually exerting toxic effects for living cells. It is clear that are suffering, but we we would like to understand if the crystals are the cause or a consequence. Does anybody have any idea or suggestion regarding that?

The differentiation medium used is composed as following:

- 1:1 of DMEM:F12 (1:1) + Neurobasal-A media

- 1% glutaMAX

- MEM non-essential amino acids solution (1X)

+ Compound E = 0,1 μM

+ L-ascorbic acid = 200 μM

+ B-27 = 1 %

+ N-2 = 0,5 %

+ ATB = 0,5 %

+ ARA (all-trans RA)= 0,1 μM

+ Purmorphamine = 1 μM

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?

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?

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!

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 :)

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?

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?

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!

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.

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.

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.

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

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 :)

#laminin #poly-D-lysine #coating #eplate #neuroncellculture #RTCA

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.

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

I am looking into protocols for culturing primary rat hippocampal neurons (no feeder layers). The standard protocol from ThermoFisher advises to replace 50% of the media every 2-3 days. However, several other sources indicate to change less of the media and less frequently. What is the best procedure for culturing previously cryopreserved neurons?

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!

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

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?

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?

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!

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?

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

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

I am looking for a b-secretase inhibitor that was used in hiPSC's neuronal culture.

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

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?

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!!

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. 

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?

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!

Does anyone have experience with the Olympus cellVivo weather chamber for monitoring neurite growth over hours/days?

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!

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?

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!

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

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!!

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)?

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

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..

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.

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?

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

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!

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?

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?

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?

Hi everyone, I have to do some experiments in another lab on campus, which is 15min walk away. I noticed the speed of neurotrophin molecules moving in axons significantly reduced to 1/3 after this 15min's physical transport (I put my culture in a enclosed foam box, where temperature and CO2 % both drop, plus mechanical disturbance) And similar phenomena were observed by other lab mates. What should I do? I read a similar thread about transporting cell lines (fill up to fullest +sealing+wrapped with towels is good for overnight), but I did all these modifications without success. Should I buy a portable incubator (any suggestions on models)? Thank you in advance for any input!

Hi everyone,

I would like to design an apoptosis induction experiment on mouse primary cortical and hippocampal neuron culture. The annexin V/PI kit seems to be the best for this. I would like to mesure in 96 well plate, but first I have to detaching the cells. The tripsin treatment seems too harmful and I am worry about the cell death, that the 90% of my culutre will be annexinV positive.

Thanks!

I am differentiating iPSCs to NPCs by overexpressing the Ngn2 transcription factor (transduced using lentiviral vector, Tet-ON promoter). After passaging, NPCs adhere, but after 1-2 days they begin to die at a high rate. NPCs that survive aggregate into clumps of varying size, but eventually detach and die.

Briefly,

iPSCs (Cultured in mTeSR) are passaged as single cells using ROCK inhibitor onto a 12 well plate coated with Matrigel (cells seeded at 6x10^5 cells/well.

Cells are fed daily for the next 5 days with DMEM/F12 + N2 supplement and doxycycline (to induce Ngn2 expression)

After 5 days, cells are passaged onto 6 well plates coated with 100ug Poly-D-Lysine and 20ug of laminin.

To passage cells, I wash with DPBS (w/o Ca or Mg) and incubate for ~5min with StemPro Accutase. after 5 min the cells lift off in a perfect circular sheet. cell are collected and spun at 1000rpm for 1min, aspirated and resuspended in DMEM/F12 + B27 supplement (and additional supplements for maturation).

I have found it difficult to generate a uniform single cell suspension, as there are large clumps that are difficult to dissociatewhile trying to not be rough on the cells, and I believe this is part of my issue.

My plate coating could also be an issue. I am currently working on optimizing the amount of each substrate to add to each well.

Any and all tips are extremely appreciated. I am new to neuron culture so anything helps.

Thanks

Brett

I am trying to improve the survivability of primary neuron cell cultures. Currently, I am getting great survival out to 20-days but would like to push my time point out to 42 days. I currently believe that the neurons are being crowded out by microglia so I am looking for ways to reduce their growth. Additionally I am curious if anyone has had any experience with 3D cell culture techniques and could provide some technical expertise and thoughts on this method compared to standard 2D cell culturing.

I have read papers that describe methods for long-term primary neuron culture but I have not seen convincing western blots showing consistent changes in any protein expression with the progress of aging.

I know that aging is a complex process, but I would like to somehow test if my long-term cultures are actually going through an aging-like process.

Hello.

i need a methionine free media for primary neuron culture, but i am not sure if methionine free DMEM it apropriated for neurons. or maybe RPMI ? i read in a a paper about methionine free Hibernate A media, but i can find that media in any company.

Thanks.

Hello everyone, I work in primary cortical neuronal culture.

Somehow my culture is contaminated and during investigation of source of contamination, we found flowing type of contamination in both NB and DMEM culture media

what type of contamination it might be

Dear all,

Which medium to use after differentiation of SHSY5Y? I used RA+BDNF and I need to use different treatment for next 2 days. Keep them in the differentiation medium or I can use standard medium for SHSY5Y cell excl. FCS, or they need some supplements?

I need to examine the effect of an agent on neuronal cells in culture. To do so, I will be pursuing neural differentiation from fibroblasts. However, whereas a number of sources indicate the necessity of co-culturing neuronal cells with astrocytes and other glial cells for production of functional neurons, my advisor maintains that it is not necessary. It is the first time I will be doing any such work and I need to avoid any technical difficulties. Can someone please help me with this?

I am trying to culture primary rat hippocampus neurons and wondered if B-27 should be sterile filtered with glutamax. I have limited experience with neuronal cultures but have run into people who tell me I should not filter B-27 while those in my current lab filter B-27 and glutamax directly into Neurobasal A media.

I currently have SH SY5Y cells from atcc and no matter how much I try to reduce clumping by pipetting up and down several times on each passage, a significant number of my cells are still clumping. There is a monolayer of cells but it appears to be the nucleus for the clumps/colonies of cells. I do have floating cells as would be expected, but they are the least of my concerns at the moment.

Media (DME/F-12 with 10% FBS and 1X PenStrep) is replaced every 4 days and cells are grown at 37 C with 5% CO2. Upon passaging of cells, a small amount of 0.25% trypsin, 0.53 mM EDTA solution is used to help adherent cells detach.

Any advice or comments on this would be appreciated. Thanks in advance.

I went through a basic protocol provided by Brewer et al., 1994

1. The media was stored according to instruction (-4 to -20 for B27, -20 for trypsin and 2-8C for neurobasal and 15C for Glutamax)

2. The hippocampus of mature rat (6W) was extracted under the sterile condition

3. The extracted hippocampus was stored in HANK and Pen/Step for 2 min

4. The hippocampus was homogenized for 15min using trypsin-EDTA 0.05 (using micropipette with blue and yellow tips). i had extrac care about buble formation

5. The trypsin was inactivated and centrifuged, the pellet was re-suspended in 500ul hank+pen

6. The cells was counted (2 * 10E6) and inoculated into the culture media (5ml neurobasal+ 2% b27+glutamax 1%) in a T25 flask (adherent + filter cap)

7. The cells were incubated at 37C and 5% Co2

* No antibiotic in culture medium / even addition of antibiotic dosen't make change. Furthermore, i didnt have contamination

* No serum in the medium

* No bFGF/ even addition of bFGF dosen't make change

Still i have some growth that i don't know what they are. I think they are not typical neural cells. In my though, the problem is that the cells are die within few days.

what went wrong?

I've performed Bradford assays in parallel with other cell viability assays. Can the level of protein in cell lysates be used as an indicator of cell viability?

Dear All,

Does someone know how we can shrink the number of days in vitro for neuronal differentiation protocols? According to the literature, it takes 40+ days (starting from human ESC/iPSC) to get more or less differentiated neurons.

Most of the protocols are employing different growth factor such as BDNF, GDNF and NT3 for neuronal maturation but it still takes plenty of time.

Any help appreciated.

Thank you in advance!

Dear All,

Does someone know how we can shrink the number of days in vitro for neuronal differentiation protocols? According to the literature, it takes 40+ days (starting from human ESC/iPSC) to get more or less differentiated neurons.

Most of the protocols are employing different growth factor such as BDNF, GDNF and NT3 for neuronal maturation but it still takes plenty of time.

Any help appreciated.

Thank you in advance!