Part 1: Items that need to be prepared before proceeding to cell plating:

1- Appropriate volume of complete NSC medium is prepared by mixing NeuroCult^® NSC Basal Medium and NeuroCult^® NSC Proliferation Supplements at a 9:1 ratio, respectively.

2- An aliquot of NeuroCult^®NCFC Serum-Free Medium without Cytokines is thawed.

3- The medium is warmed up in a 37ºC water bath.

4- Stock solutions of epidermal growth factor (EGF), basic fibroblastic growth factor (b-FGF) at the concentration of 10 µg/ml and heparin at 0.2% are prepared ahead.

5- Depending on the experiment size, several 35mm tissue culture dishes are needed to plate the cells and one 150-200cm plastic Petri dish is also needed to hold the duplicate 35mm dishes and a third 35mm dish for water.

Part2: Cell preparation:

Depending on your experiment, cells can be prepared from an adult or embryonic source (primary dissociated tissue or dissociated neurospheres). Dissect tissues from adult/embryonic mouse central nervous system (CNS) or dissociate adult/embryonic-derived neurospheres as described before ^1,2 and then:

1- The single cell suspension is passed through a 40-µm-size mesh filter so as to remove non-dissociated clumps.

2- 10µl of the cell suspension is mixed with 90µl of Trypan blue to perform a cell count. Note: Other appropriate cell dilutions can also be used.

3- If using primary embryonic or adult derived neural cells, dilute the cell suspension to a concentration of 6.510⁵ cells/ml in complete NSC medium. If using cells from dissociated neurospheres derived from adult or embryonic neural cells, dilute the cell suspension to a concentration of 2.2 10⁵ cells/ml in complete NSC medium.

Part2: Plating cells in semi-solid N-CFCA medium:

1- The appropriate volume of the following components is mixed in order, depending on the number of replicates. Here we mix the amount needed for two replicates or duplicate dishes. For additional numbers of replicates, please refer to table 1.

• 1.7 ml of NeuroCult® NCFC Serum-Free Medium without Cytokines

• 330 µl of NeuroCult® NSC Proliferation Supplements

• 6.6 µl of EGF (10µg/ml)

• 32 µl of Penicillin/Streptomycin

• 3.3 µl of b-FGF (10µg/ml) required only if culturing cells derived from adult neural cells.

• 3.3 µl of heparin (0.2%) required only if culturing cells derived from adult neural cells.

• 25 µl of cell suspension (of 2.2 10⁵ cells/ml cells from dissociated neurospheres or 6.5 10⁵ cells/ml primary cells from dissociated CNS tissue). Note: The final cell numbers plated should be about 2500 cells per 35 mm culture dish for neurosphere derived cells and 7500 cells per 35 mm culture dish for primary cells. In some cases, the final cell plating density has to be adjusted by performing a cell titration experiment. For statistical analyses, the total number of colonies detected after 21 days of culture should be within the range of at least 50 – 150 colonies.

2- The medium containing the cells is mixed gently and then 1.3 ml of cold Collagen solution is transferred to the cell suspension and mixed well by gentle pipetting to avoid introducing any bubbles.

3- The mixed solution is dispensed to the center of each 35mm culture dish (~1.5 ml/dish) and the dishes are tipped gently using a circular motion to let the mixture spread evenly over the surface of the dish. .

4- The duplicate 35 mm culture dishes are placed into a 100 mm Petri plate. The lid of new 35 mm dish is removed and the open dish is also placed in the same 100 mm Petri dish. Sterile water is added to this open 35 mm dish to maintain optimal humidity during the incubation period. Square bioassay plates (245 mm) are used when more replicate 35 mm dishes have been set up. Again, include 2 or 3 open 35 mm dishes containing sterile water.

5- The plates are transferred to an incubator set at 37°C, 5% CO2 and 95% humidity. The collagen congeals by increasing temperature and gel formation will occur within approximately one hour. The cultures should not be disturbed during this time.

6- Cultured cells are incubated for 21 days (differences in colony size can be clearly distinguished after 21 days).

Part4: Preparing replenishment medium and feeding the culture:

As N-CFCA cultures are incubated for an extended period of time (21 days), cultures should be fed with the appropriate complete NeuroCult^® replenishment medium prepared fresh as follows:

• 4.5ml of NeuroCult^® NSC Basal Medium is mixed with 0.5ml of NeuroCult® NSC Proliferation Supplements and then the growth factors is added:

• 250 μL of 10μg/ml EGF (to give a final concentration of 0.5μg/ml EGF)

• 125 μL of 10μg/mL b-FGF (to give a final concentration of 0.25μg/ml b-FGF) required only if culturing cells derived from adult neural cells.

• 125 μL of 0.2% Heparin, required only if culturing cells derived from adult neural cells.

• 60 μL of the appropriate complete NeuroCult® Replenishment Medium (for embryonic or adult cells) is added into the center of each NCFC Assay dish once every 7 days during the entire NCFC Assay culture incubation (21 days).

Part5: Scoring N-CFC Assay derived colonies and calculating the frequency of bona fide NSCs and neural progenitor cells:

• Each 35 mm culture dish is placed on a gridded scoring dish with the grid size of 2.0 mm 2.0 mm and then both dishes are placed on the microscope stage.

• Using low power (2.5X - 5X) objective lens, each dish is scanned and the colonies are scored based on their sizes.

• Colonies are classified into two major categories:

1. Less than 2 mm diameter

2. ≥2 mm diameter

Representative Results:

As in neurosphere assay, cells plated in N-CFC assay start to proliferate and make small colonies of cells within 3 - 7 days after plating (Figure 1). In two weeks time, colonies of different sizes can be distinguished. While the majority of colonies tend to stop growing after 14 days, some colonies continue to increase in size. By day 21, colonies can be classified into one of the four categories: 1) less than 0.5 mm diameter, 2) 0.5 - 1 mm diameter, 3) 1 - 2 mm diameter and 4) ≥2mm in diameter. Practically, colonies smaller than 2 mm diameter are referred to as progenitor derived (Figure 2) and colonies ≥2mm in diameter are referred to as NSC derived (Figure 3). The number of colonies ≥2mm in diameter per total cells plated, represents the frequency of the actual bona fide neural stem cells with long-term self-renewal and multi-potential capabilities. The total neurosphere forming frequency in a particular cell population after 7-8 day in a parallel NSA experiment has been estimated to be similar to the total colony forming frequency of the same cell population after 21 days in a N-CFCA experiment. The N-CFCA however, provides a more permissive condition so as each cell can show its full proliferative potential.

Table 1. Components of complete N-CFC assay culture.

Component	2 replicates	3 replicates	4 replicates
NeuroCult® NCFC Serum-Free Medium without Cytokines	1700	2550	3400
NeuroCult® NSC Proliferation Supplements	330	495	660
EGF (10 μg/mL)	6.6	9.9	13.2
bFGF (10 μg/mL), only for adult cells	3.3	4.95	6.6
Heparin Solution (0.2%), only for adult cells	3.3	4.95	6.6
Penicillin/Streptomycin (1:100)	32	48	64
Cells at: 2.2 x 105 cultured cells/mL OR 6.5 x 105 primary cells/mL	25	37.5	50
Collagen Solution	1300	1950	2600