The aim of this video article is to demonstrate how to quantify the frequency of bonafide neural stem cells in a mixed population of neural precursor cells using the neuro colony forming cell assay. This assay can be used for primary or cultured neural precursor cells from adult or embryonic CNS harvested. Single cells are plated at an appropriate density and then incubated for three weeks in a 37 degree incubator.
The frequency of neural progenitor and bonafide neural stem cells is calculated respectively by counting the number of colonies that are less than two millimeters and greater than or equal To two millimeters. Using the neuros scale assay To determine the frequency of bonafide neural stem cells in a mixed neural cell population, overestimates the number of neuro stem cells because there's not a one-to-one relationship between neuro sphere formation and neuro stem cells. We developed the neural colony for mix cell assay in our lab.
To overcome this limitation, this assay can easily discriminate bonafide neural stem cells from pro gender cells based on the long term proliferation potential. Today we show you how to use this semi-solid collagen based assay to enumerate the frequency of neural stem cells. The cells prepared from dissociated neurospheres from E 14 mile screen.
Okay, let's get started. For this assay, we need neural cult neural stem cell proliferation, supplement basal medium neural colony forming cell assay, serum free medium, and collagen pipette, one ml of complete neural stem cell medium into a 15 mil falcon tube. Take as much volume from the cell suspension you have already prepared and pass through a 40 micron mesh filter in order to reach a concentration of 2.2 times 10 to the five cells per mil de plate cells in duplicate.
In 2 35 millimeter culture dishes. Mix 1.7 mil of neuro cult neural colony forming cell assay serum free medium without cytokines with three 30 microliter of neuro cult neural stem cell proliferation supplement. Then add 6.6 microliter of EGF and 32 microliters of penicillin streptomycin.
Add 25 microliter of cell suspension to the prepared medium. Add 1.3 mil of cold collagen solution to the cell suspension and mix well, but gently to avoid introducing air bubbles. Then dispense the mixed solution to the center of each culture dish, approximately 1.5 mil per dish and let the mixture spread evenly over the surface of the dish.
Three ml of sterile water is added to an open 35 millimeter dish placed alongside the culture dishes in the large Petri plate. This helps maintain optimal humidity during the incubation period. Label the plate and transfer to an incubator set at 37 degrees Celsius, 5%carbon dioxide, and 95%humidity.
It is important not to disturb the culture during the first hours So that the collagen can congeal to feed the neural colony forming cell Assay culture. Take 200 microliter of complete neural stem cell medium and add 10 microliters of EGF and a concentration Of 10 micrograms per mil. Add 60 microliter of The complete neuroco replenishment medium to the center of each dish once every seven days during the entire incubation period.
Each culture dish is placed on a grided Scoring dish of grid size, two millimeter by two millimeter, and then placed on the microscope stage using low power objective lens. Each dish is scanned and colonies are scored based on their size. Here are representative results of the assay at the end of the first week.
At this time point, neural stem and progenitor cell derived colonies are indistinguishable. After three weeks, colonies can be classified into two major categories, those less than two millimeter in diameter, which are progenitor cell derived, and those greater than or equal to two millimeter in diameter, which are stem Cell derived. The colonies that are greater than or equal to two millimeters in diameter fulfill all the functional criteria for bonafide neural stem cell In The colony.
Forming Cell assay plating cells with an appropriate density is very important. Having too few colonies keeps the rare neural stem cell colonies under detection levels. On the other hand, too many colonies will result in overcrowding and deficient of gross medium components.
Thank you for watching and good luck with your experiments.
