Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

The overall goal of this procedure is to follow the cell cycle progression of sorted neural stem cells and their progeny isolated from the subventricular zone of transgenic fluorescence ubiquitination cell cycle known as FCI mice. This is accomplished by first dissecting the sub ventricular zone of transgenic adult mouse brains. The second step is to dissociate the neuronal tissue into a single cell suspension using a papayan treatment.

Next, the different neurogenic cell populations of the subventricular zone are labeled using antibodies in order to identify quiescence neural stem cells, activated neural stem cells, transit amplifying cells, and immature as well as migrating neuroblast. The final step is to use facts to sort the cells directly into fresh culture medium before plating them on poly D lysine coated plates. Ultimately continuous time-lapse video microscopy is used to study the cell cycle progression of the plated cells as the various transgenic cells fluoresce at different time points during their cell cycle.

Combining the cell sorting technique with the Fuji technology allows the visualization and the isolation of the different cell populations from the sub ventricular zone, enabling the study of the properties and dynamics in the adult movement brain. This method can help answer key questions in the context of aging and brain pathologies The day before the experiment. At about 200 microliters of a 10 microgram per milliliter poly D lysine solution to each well of a sterile black walled 96 well plate to coat the bottom of the wells cover and incubate the plate overnight at 37 degrees Celsius the next day.

Remove the poly D lysine solution and rinse the wells three times with sterile distilled water before allowing the plate to dry in the hood under laminar flow for at least two hours. If not used immediately. Store the dried coated plate at four degrees Celsius for up to seven days.

Next, prepare the Papain dissociation solution and sterilize it by passing the mixture through a 0.2 micron filter. Prewarm the solution to 37 degrees Celsius before it is used. Also, prepare a protease inhibitor solution by adding 0.7 milligrams per milliliter of trypsin in inhibitor type two to TM F 12 medium sterile.

Filter the solution using a 0.2 micron filter and prewarm the solution to 37 degrees Celsius before it is used. Place the freshly dissected sub ventricular zone from an adult fucci red mouse brain into a Petri dish containing 0.6%Glucose in PBS mince the tissue until no large pieces remain. Then transfer everything into a 15 milliliter tube and centrifuge at 200 times G for five minutes.

Once the tissue has been pelleted, discard the snat and resus. Suspend the tissue in one milliliter of prewarm papain, supplemented with 0.01 milligrams per milliliter of dna.One. Incubate the mixture for 10 minutes in a water bath at 37 degrees Celsius.

Then centrifuge again at 200 times G for five minutes and discard the snat. Add one milliliter of the Prewarm OVO MOID solution to stop the enzymatic digestion. Next, use a P 1000 micro pipette tip to gently pipette up and down 20 times to mechanically dissociate the minced tissue into a single cell suspension while pipetting.

Avoid introducing any air bubbles into the mixture which would cause extra stress to the cells. Pass the cell suspension through a sterile 20 micron filter in a new 15 milliliter tube. Wash the cell filter with two milliliters of PBS containing 0.15%BSA to avoid losing cells.

Then centrifuge the cell suspension at 200 times G for 10 minutes. Discard the supernatant. Perform the fact staining by first suspending the cells from each mouse into 100 microliters of PBS containing 0.15%BSA.

Use one 10th of the cells from one of the mice to prepare each of the four controls as described in the accompanying text protocol. Add the primary antibodies listed here to each of the tubes used for cell sorting and incubate the mixtures for 20 minutes in the dark at four degrees Celsius. Following incubation with the primary antibodies, wash the cells with one milliliter of PBS containing 0.15%BS, A, and then centrifuge the cells at 200 times G for 10 minutes.

Re suspend the cells in 200 microliters of PBS containing 0.15%BSA per brain and transfer them into appropriate effects. Sorting tubes. Place the cell sorting tubes on ice and add a vital dye such as hooked 3 3 2 5 8 stain at two micrograms per milliliter to the cells immediately prior to cell sorting to discriminate live from dead cells.

Next, run the cells in the negative control tube through the facts and select the cells using side scatter and forward scatter parameters. Exclude the dead cells by gating only the hooks to negative fraction and doublets by selecting the pulse with negative fraction. Run single color controls and adjust the photo multiplier tube voltages if necessary.

Perform color compensation in the compensation window of the software. Run the three fluorescence minus one controls and draw the sorting gates. Once all the gates are set up, sort the labeled cells directly into 100 microliters of culture.

Medium plate the freshly sorted cells at a density of 1000 to 3000 cells per well onto the poly de lycine coated. 96 well plates with 300 microliters of culture medium. Incubate the culture plates at 37 degrees Celsius and 5%carbon dioxide for at least one hour.

To allow for cell adhesion, prepare a confocal laser scanning microscope for imaging by preheating the attached temperature controlled chamber to 37 degrees Celsius under a 5%carbon dioxide atmosphere. Position the 96 well plate inside the prewarm microscope chamber and bring the first well into focus. Then open the imaging software and right click in the main window to select acquire acquisition controls and DEAC acquisition.

In order to open the TI pad and select the plan A PO VC 20 XDIC objective next, click on acquire acquisition controls and then open the time-lapse options. Set the center of each well as a stage position and select the large image option to seven by seven millimeter squared. Observe a mosaic image around the center of each.

Well set the overlap for the large mosaic image to 5%and set the frequency so that pictures are taken every 20 minutes for 24 hours. Under a one plus settings, select the photo multiplier tube voltage level for each fluorescence listed in the menu bar. Select to acquire images using a high speed resonance scanner at a five 12 by five 12 pixels Format and use DIC to visualize the cells.

Then select a folder to save the data files. Select the run now button on the ND acquisition window to begin acquisition. Follow along with the computer work for the duration of at least one loop to be sure that everything is working properly.

Fuji red mice were used to follow the G one phase with red fluorescence using time-lapse video microscopy. The isolated cells are red fluorescent during the first growth phase and our color list during synthesis, the second growth phase and mitosis. Lex positive eeg FR positive and eeg.

FR positive cells from young adult and middle aged mice were plated and tracked via microscopy. The S and G two M phase length showed no difference between Lex positive EGFR positive and EG FFR positive cells either in young or middle-aged mice. However, the length of the next growth phase from the first division until the red fluorescent switches off was found to be longer in the older cells due to the increase in G one phase length.

Neurospheres obtained five days after plating are smaller in Lex positive EGFR positive cells obtained from the older mice. The entire protocol should not exceed three hours for dissection and fax preparation and another three hours for cell sorting when working with 12 mice. Keep in mind our working with too many mice on the same day will lead to an increased cell sorting duration and possibly resulting in an increased cell death or cell differentiation.