Are u sure the DNAse is working? Or perhaps u did not removed the meninges completely?

My guess is either DNA or collagen.  Probably collagen, use one of the collagenase enzymes to break it up.

Hi Hao, from what you describe, it sounds like you are dealing with DNA - despite the use of DNase. Checking your DNase would be the first thing I'd recommend you do. I would also recommend to stay away from increasing the concentration of your trypsin, as this may be bad for the overall health of your cells. You could also try decreasing the incubation time in trypsin and see if this helps. Good luck!

Dear Hao, I do not have much to add to the comments above. From my experience with preparing cortical neurons and motoneurons from mouse embryos, the cloud of white insoluble material you describe is very likely made of DNA. The higher the trpysin, the more DNA, to an extent that may exceed the working capacity of the DNAse. You might use more DNAseI or get another batch with certified activity.

Trypsin is very cytotoxic and readily cause cellular breakdown. A few things you may want to try to prevent cellular breakdown during trypsin digest and increase the yield of cell isolation:

(1) reduce trypsin concentration 2-5 times instead of increasing it,

(2) make sure to use a buffer without Ca2+ and Mg2+ (e.g. PBS-/- or much more physiological HBSS-/-) during the dissection and digestion steps. Ca2+ and Mg2+ are cofactors for trypsin and make it too toxic; removing these metal ions keeps the activity low but still more than enough for releasing cells,

(3) Addition of 10mM HEPES, pH 7.4 (for instance from a 1M sterile stock form Invitrogen/LifeTechnologies) during the dissection and digestion steps helps preventing acidification of the medium as some cells die

(4) reduce the speed of the magnetic stirrer during the digestion steps at 37°C (60-100 rpm is enough)

(5) perform several rounds of digestion, for instance 4-6 rounds of 5 min each. After each 5 min period, let the pieces of minced tissues settle down, harvest the supernatant, supplement with 5-10% FBS and keep it at 4 degrees to prevent trypsin. At the end of the process, pool the successive fractions and pellet the cells by gentle centrifugation (10 min at 300g) before resuspending the cells in your medium.

I hope it helps.

All the best with your experiments.

Olivier

好像很有道理的样子

本来protocol上是先加DNase 然后trypsin，一起水浴了30分钟

但我们是水浴之后才加，而且过不久就马上离心倒掉了.......DNase还没起作用

I agree with the above.  I think that you are tearing up too many cells and that the DNA from the dead cells is too much for the DNAse in your solution to deal with.  for best results, dissociate CNS tissue slowly and gently. 

Titration of enzymes and the optimization of buffers are key to achieve a perfect single cell suspension.

If possible ( and depending on your cell type of interest of course) use young mice around birth. The first step, the dissociation is really crucial. If somthing goes wrong here the rest of the experiment won't work well.

If you like complete kits with detailed optimized procotols, you could try the "Neural Tissue Dissociation Kit-Postnatal Neurons" (which works also fine for neurons from embryonic tissue but also even from early postnatal tissue ). For ultimate standardization you can even combine the kit with a dissociation instrument, the gentleMACS, which can incubate and dissociate mechanically for you.

By using just culture selection e.g. the supplement B27 or Neuro Brew-21 on a mixed culture, you will not get a pure neuron culture from neonatal or postnatal  animals. Our experiments have shown that these supplements actually support also the growth of glia. An enrichment step is crucial for highest purity.

For the magnetic isolation of cells , you could next use the "Retinal Ganglion Cell Isolation Kit, rat" to get only this one specific kind of neurons or the "Neuron Isolation Kit, mouse" to get rid of all other cell types and end up with untouched neurons.

Good luck !! :-)

Probably collagenase is much less offensive than trypsin. So, use collagenase and jaluronidase. For long time and after carefully dissecting the tissue in

During the development of the Neural Tissue Dissociation Kits we tested different enzymes and many different concentrations and buffers. Our kit with papain shows the best performance, even better than the trypsin-based kit. It is also important to use always the number of activity units (not volume or weight of the enzyme). Our kit always contain the same number of units, so it's a standardized protocol that gives reliable and reproducible results. A huge number of labs worldwide has been using our different protocols for neural tissue. Check out the publications by searching e.g. Highwire  "Miltenyi AND "dissociation kit" ".

As others have mentioned, probably DNA, or maybe even fat.  At this age, I don't think myelin is a huge problem. 

I have isolated primary cultures from neonatal mouse cortex and have used the Miltenyi Biotek kit (you'd need their machine to dissociate the tissue though so it might be expensive) as well as trituration with flame polished pasteur pipets.  Both have worked well. 

For trituration, I generally use 0.25% Trypsin for 5-10min, inactivate the trypsin with 10% FBS, and then break up the tissue with decreasing sizes of flame polished pipets until I get a single cell suspension (checked with hemacytometer).  Method seems to work well for neurospheres as I can get spheres to stay in culture and proliferate for over 10 weeks and spheres can be re-dissociated and form a monolayer of neuronal and glial cells on adherently coated surfaces.

Hi, the Neural Tissue Dissociation Kit-P (or -T) as well as the Neural Tissue Dissociation Kit- Postnatal Neurons contain both, a protocol for manual dissociation with classical pasteur pipettes and a protocol for dissociation using one of the gentleMACS Dissociators for ultimate standardization or large numbers of animals. Data sheets are available for download:

http://www.miltenyibiotec.com/en/products-and-services/macs-sample-preparation/sample-dissociation/tissue-dissociation-kits/neural-tissue-dissociation-kits.aspx

From my experience, the "sticky white phenomenon" more likely occurs when using trypsin rather than papain. The Neural Tissue Dissociation Kit-Postnatal Neurons is papain-based.

We had tested also collagenase and hyaluronidase (both very expensive) during our development phase, but papain and trypsin had shown better results.