The aim of this video protocol is to demonstrate the generation and enrichment of neuronal progenitor cells from differentiating neuro stem cell progeny using flow cytometry. This is achieved by first isolating and expanding neuros stem cells using the neuros sphere assay. The resulting cells are then differentiated through a two-step method called the neuroblast assay.
Finally, using flow cytometry, distinct populations of cells can be identified and sorted based on their physical and fluorescent property. In this protocol, we'll introduce novel methods for both the differentiation of neuro stem cells, which is called a neuroblast assay, as well as for the purification of the resulting immature neural progeny using flow cytometry. The Neuroblast assay has a proliferation phase, which takes about three to four days, and a differentiation phase that takes about four to five days.
During the differentiation stage, a mono layer of cells formed, which is composed of astrocytes underneath with clusters of immature neurons on top. When the differentiating neuros stem cell progeny are dissociated into single cells and then run through the flow cytometry machine, a sharp difference can be distinguished between the neuro neuro progenitors and the rest of the cells in terms of their size and internal complexity. Gating the small cell population, which has low forward and side scattered properties, highly enriched neuronal cells can be isolated to achieve an even more homogenous neural cell population.
The dissociated neuro blood assay culture can be asain for PSA and cam, and the positive cells can be gated and sorted from the small cell population. Okay, let's get started. Neural stem cells are isolated and expanded from mouse embryonic.
Day 14 ganglionic eminence using the neuros sphere assay as appears in a separate video protocol, which can be found at the address on the screen. Ensure that the spheres have reached a diameter of 150 to 200 microns. Collect the spheres and transfer to an appropriate sized sterile tube.
Spin the tube at 800 RPM or 110 G for five minutes. Then remove the supernatant and resuspend the spheres in one mil of prewarm trypsin EDTA solution. Incubate the cells in a 37 degree water bath for two to three minutes.
Add an equal volume of trypsin inhibitor to the cells and mix well to quench the tryin activity and achieve a single cell suspension. Transfer the cells to a smaller sized tube and spin again at 110 G for five minutes. Remove the S natin and resuspend the cells in one to two mils.
Complete neural stem cell medium. Take 10 microliters of the cell suspension and mix well with 90 microliters of triam blue and perform a cell count. To prepare the growth culture medium, add fetal calf serum to a final concentration of 5%in complete neural stem Cell medium.
Then add the cells to the culture medium To reach a density of two to 300, 000 cells per ml. Now add EGF at a final concentration of 20 nanograms per ML and BFGF at a final concentration of 10 nanograms per ml. Mix the cell suspension thoroughly to make it homogenous and plate 20 mils per T 80 flask.
Place the flasks in the incubator at 37 degrees and 5%CO2 for three to four days. The neural stem cell progeny will attach to the substrate and proliferate as a monolith three to four days after the initial plating, the culture will have grown 90 to 95%confluent and is ready now to have the medium switched to growth factor free medium for differentiation. Now prepare fresh neural stem cell medium supplemented with 5%FCS without any growth factors.
Remove the medium from each flask and replace with the growth factor free medium. Incubate the flasks for four to five additional days. During this time, neuronal progenitor cells will actively divide and generate colonies of immature neuronal cells, which will appear on top of an astrocytic monolayer.
To prepare the cells for flow cytometry, first, remove the medium from the flask on day five of differentiation stage. Then to remove fetal cast serum from the culture, add four mils of PBS swirl gently for about 30 seconds and remove then a sufficient amount of prewarm tripsin. EDTA is added to the flask to cover the cell monolayer.
Place the flask in a 37 degree incubator for one to two minutes, then smack the flask two to three times to ensure the cells become detached. Now an equal volume of tripsin inhibitors is added to the flask to quench the tryin activity. The cell suspension is gently pipetted up and down to ensure inactivation of trypsin and to achieve a homogenous single cell suspension.
Transfer the contents of the flask to an appropriate sized tube and spin at 110 G for five minutes. Remove the supernatant, then resuspend The cells in an appropriate volume of neuro stem cell medium and perform a cell count as described before the single cells harvested from the neuroblast assay. Culture are divided into four groups, cells alone, cells plus pi, isotype control and PSA in cam.
Add medium to the cells alone and cells plus PI tubes so that each reaches a total volume of one mil. Make sure their densities do not exceed two to 3 million cells per ML to avoid stream blockage in the flow cytometry machine centrifuge, the isotype control and PSA and cam tubes. Remove the supra natin from both tubes and resuspend the cells in 100 microliters of complete neuro stem cell media.
Following Vendor instructions, add the appropriate amount of PE conjugated isotype control and PE conjugated anti PSA and cam antibodies to their respective tubes. Mix the samples gently and Incubate for 10 minutes at room temperature in the dark. Centrifuge the samples and remove the SUP natin from both tubes and resuspend the cells in one mil of complete neuro stem cell medium.
Repeat this procedure two to three times to remove excess unbound antibodies after the last wash. Resuspend each sample in One mil of complete medium. Add propidium Iodide at a concentration of one microliter per ml to each sample.
With the exception of the cells Alone group, Prepare three 15 ml tubes with one mil of complete neuro stem cell medium for collection of the different cell populations by flow cytometry Before beginning Fluorescence. Activated cell sorting machine must be adjusted for laser alignment, voltage parameters and proper functioning by the facility expert technician. Once these adjustments have been made, run the cell suspension from the cells alone group through the fax machine.
First, the acquired events are plotted based on their forward and side scattered properties to distinguish different cell populations. To do so, voltage should be adjusted so the events can be seen in the flow cytometry plot. Now run the cells plus PI sample to exclude cell clumps and doublets.
The events are plotted based on forward scatter area versus forward scatter pulse width and the single cell population is gated as population one. Fine tune the gate to exclude events with high forward Scatter pulse width. Then to further exclude clumps and doublets.
The population one cells are plotted based on side scatter area versus side scatter pulse width, and this time the single cells are gated as population two. Events with high Side scatter pulse width are excluded to exclude dead Or damaged cells. The single cells of population two are plotted based on PI reactivity versus forward scatter area, And the single live cell population is gated.
Now single live cells are plotted based On forward scatter area versus side scatter area. At this stage, small and large cell populations are gated. The small population has forward scatter, low side scatter low properties, and is named here population three.
The large population has forwarded scatter high side scatter high properties and is named here population four to select PSA and CAM positive cells First. The forward scatter low side scatter low populations from cells plus PI and isotype control groups are plotted based on PE immunoreactivity versus forward scatter area, and the negative cells are gated as population five. Then the PSA and CAM stain sample is run through the machine and the positive events gated as population six.
The 15 Mil sterile tissue culture tubes that contain one mil of neuro stem cell medium are placed in the machine to collect sorted cell populations. Then gated cell populations Of interest are selected and sorted after the Cells have been sorted. Return to the tissue culture hood and wash the tubes to free up any sorted cells that have stuck to the sides.
Then spin the tubes at 1200 RPM or 240 G for five minutes, discard the sup nain and resuspend the cells in an appropriate amount of complete neuro stem Cell medium. Then perform a cell count as shown before to plate the cells. First, prepare a sufficient amount of complete neuro stem cell medium, supplemented with 5%FCS and 20 nanogram per ml.
Human recombinant BMP four. Mix the medium thoroughly and distribute into newly labeled tubes corresponding to the Different sorted cell populations. Add the sorted cells to the corresponding tube to reach a density of about a hundred to 150, 000 cells per ml.
Now remove the medium from the polio coated 96 well plate mix the cell suspensions and add 200 to 250 microliters per well label the plate and place it in the incubator at 37 degrees And 5%CO2. This is an example of a neuroblast assay culture from E 14 mouse neuro stem cells. On day four of proliferation stage arrowheads indicate the flat astrocytic cells underneath and arrows indicate the round neuronal progenitor cells on top.
This is an example of a neuroblast assay culture five days after switching to growth factor three medium. As you can see, there are clusters of neuronal progenitor cells growing on top of an astrocytic monolith. Here's an example of an immunofluorescent staining of the same culture.
Note the cluster of beta three tubulin stained immature neuronal cells that are on top of GFAP expressing astrocytes. This is an example of cells that were sorted from the forward scatter low side scatter low population of which the majority are expressing the neuronal progenitor marker beta three tubulin. These are cells that were sorted from the forward scatter high side scatter high population, the majority of which are ggl fibrillary acidic protein expressing astrocytes.
These are the PSA and CAM positive sorted cells from the forward scatter, low side, scatter low population, and almost all are beta three tubulin expressing neurons sorted neuronal progenitor cells can survive in culture and mature into GABAergic dark 32 expressing medium spining neurons. We've just shown you How to generate and purify immature normal cells from differentiating neuro stem cell progeny to have a high quality nerve loss asay culture, we recommend not to let the sphere grow too large. Large spheres are accompanied with more cell deaths and less neurogenic abilities.
Do not trypsin the spheres for more than two to three minutes over trypsin causes damage to the cells and decreases their neurogenic ency. Also, do not let the perforating more layer become over confluent. This might interfere with the normal differentiation process, so always switch medium when the culture reaches about 90%co fluency and when using pro cytometry, it is highly advised to use antibiotics in your medium to prevent contamination.
Thank you for watching and good luck with your experiments.
