Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system. Hundreds of neuronal glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules or molecular determinants through which it can be identified and characterized.

Currently, robust cell identification and separation technologies require single cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentle max associator when used in combination with trypsin or pepane based dissociation kits can effectively and gently dissociate brain tissue while preserving antigen epitopes in limiting cell loss. Once generated single cell suspensions can be treated with monoclonal conjugates like anti prominent one microbeads, which identify neuro progenitors or purified further using myelin removal beads.

Hi, my name is Sandra PeNAT. I'm a project manager for neuroscience products at Mil Biotech here in baggage Gabert Germany. Today I will show you how to prepare a single cell suspension from mouse brain tissue using the genix dis associator.

The genix dis associator is a device that can dissociate a variety of tissue types in a closed sterile environment, and so it eliminates the need for tedious manual mechanical dissociation. So let's get started. Depending on the antigen epitope of interest in subsequent applications use either the papain based neural tissue dissociation kit or the trypsin based neural tissue dissociation kit.

For today's experiment, we prefer the neuro tissue dissociation kit P as we would like to isolate prominent positive cells using anti prominent one microbeads. Prominent one is neither sensitive to pepane nor to trypsin, and in such a case, the neuro tissue dissociation kit P is the better choice. Before the protocol is initiated, the reagents and solutions must be prepared for this kit.

Have on hand your own stock of Hank's balance salt solution HBSS without calcium ions and magnesium ions HBSS with calcium and magnesium ions and beam me capto. Ethanol begin by preparing solution two solution four and subsequently enzyme mix one. First, add betta me capto ethanol to solution two to a final concentration of 0.067 millimolar.

The solution is now stable for up to one month when stored at four degrees Celsius. Next, dissolve the lyophilized powder and a vial labeled solution.Four. Using 0.7 milliliter of storage buffer mix gently and do not vortex.

Now prepare enzyme mix one by pipetting 1900 microliters of solution two and 50 microliters of solution one in into a C tube. This is enough solution to dissociate 400 milligrams of tissue, so if more will be dissociated, prepare additional enzyme mix one. A maximum of 1600 milligrams of brain tissue can be processed in a C tube.

The enzyme mix one in the C tube is then incubated at 37 degrees Celsius for 10 to 15 minutes. To begin the dissociation pipette one milliliter of HBSS without calcium and magnesium ions into the tube. Set the tube on a balance, add the brain tissue and record its mass.

This P four mouse brain weighs about 0.2 grams. An adult brain would weigh about 0.4 grams for all tissue samples less than 400 milligrams. The solution volumes we will use today are adequate.

If your tissue weighs more than 400 milligrams than scale up all reagent volumes accordingly, up to 1600 milligram tissue can be processed in one C tube. Transfer the mouse brain into the prepared C tube containing 1, 950 microliters of preheated and enzyme. Mix one and seal the cap tightly.

Now place the C tube onto the gen max associator with the cap down and make sure that the tissue is located in the area of the stator. Select the program MCO brain O one and run the program. This is the first of three programs you will run on the dissociation when the program finishes.

Remove the C tube and incubate the sample for 15 minutes at 37 degrees Celsius under slow continuous rotation. Using the max mix tube rotator, the running mode of the device corresponds to four RPM. After the first incubation, put the tube back onto the dissociation upside down and run program M brain O2.During the next association, prepare 30 microliters of enzyme mix two per 400 milligrams of tissue by adding 20 microliters of solution, three to 10 microliters of solution four.

When the second associator cycle has come to an end at 30 microliters of enzyme mix two into the C tube directly through the septum sealed opening in the center of the cap. To ensure sterility, invert the C tube gently to mix and do not vortex. Then put the C tube back on the max mix tube rotator and incubate the sample for 10 minutes at 37 degrees Celsius with four RPM mixing.

Then the CTU is returned onto the gentle max dis associator for the third and last time, and the program M brain oh three is run. When the third program is completed, put the C tubes on the rotator and incubate for 10 more minutes at 37 degrees Celsius. Now centrifuge the sample briefly to collect the cells at the bottom of the tube.

We are now ready to filter the cells. Choose a cell strainer according to the size of your cell type of interest. If the cells you want to collect are greater than 30 micrometers in diameter such as kinji cells, the pore size must be large enough to pass cell bodies of this diameter.

For prominent one positive cells, a 30 micrometer filter will work. Remove the dissociated tissue from the C tube using 1000 microliter pipette tips that fit through the septum sealed opening in the C tube cap. Apply the cell suspension to the pres separation filter placed on a 15 milliliter collection tube.

For larger sample sizes of 50 milliliter tube may be required. Allow the cell suspension to run through the filter. Then wash the filter by adding 10 milliliters of HBSS solution with calcium and magnesium ions.

When working with tissue quantities greater than 400 milligrams, use up to 30 milliliters of HBSS with calcium and magnesium ions for the washing of the filter. Now pellet the cells in the collection tube by centrifugation at 300 Gs for 10 minutes at room temperature, aspirate the supernat completely and resuspend the cell pellet in your buffer or medium of choice. At this point, you may choose to remove any myelin debris as it can impair cell isolation and antibody staining.

To remove myelin debris, we suggest using myelin removal beads. Otherwise, the cells are ready for further application. This dot plot based on propidium iodide staining indicates that 97%of cells present in a single cell suspension from home mouse sprain like the suspension generated in this video are viable.

These cells can be further purified using anti prominent microbeads to yield neuronal progenitors, which when cultured in max neuro medium, supplemented with B 27 plus can form neurospheres in these dot plots. The benefit of treating suspensions with the myelin removal beads is observed. A significantly higher percentage of prominent one expressing neuronal progenitors is obtained by max separation following myelin depletion from a single cell suspension.

I have just shown you how to prepare single cell suspension from mouse brain tissue using the genix dissociation and the neural tissue dissociation kit. When doing this procedure, it's important to have all solutions prepared appropriately for the right choice of the neural tissue dissociation kit, T or P.Please refer to the NDK application note or data sheet. Using this method, embryonic as well as the D tissue sections or whole brain can be processed under sterile conditions in a time saving and standardized manner.

Thank you for watching and good luck for your experiments.