Primary Striatal Neuronal Culture - Drugs of Abuse: Neurological Reviews and Protocols - page 372

3.3. Cell Trituration, Suspension, and Plating

1. Remove the dish to culture hood. Cut striata into approx 1-mm square pieces in
the dish. Using a pipet fi ller/dispenser and 10-mL pipet, aspirate the pieces of
striata with the 1X PBS into a 15-mL screw cap centrifuge tube. Fill the tube
with 1X PBS up to 10 mL.

2. Add 1 mL of 2.5% trypsin (which should be prewarmed to 37°C in a water
bath) to the tube to achieve a fi nal concentration of approx 0.25% for proteolytic
digestion of striatal tissues ( see Note 3 ). Place the tube in a 37°C water bath for
10 min, periodically and gently inverting.

3. Centrifuge for 5 min at 100 g .

4. Remove supernatant (debris) from the tube with a sterile Pasteur pipet and add
10 mL of trituration solution. Trituration solution is used to inactivate trypsin
and allow for mechanical dissociation of neural tissue to produce a fi nal cell
suspension. DNase in trituration solution is used to prevent cell clumping caused
by release of DNA from cells damaged in the cell suspension procedures.

5. Triturate striata by gently aspirating and expelling striata in trituration solution
10-15 times with a sterile Pasteur pipet, until the tissue breaks into smaller
pieces. Disperse the small clumps of tissue into single individual cells by
repeating the trituration 8-10 times with a fl ame-narrowed (polished) Pasteur
pipet, the bore of which is narrowed to about one third (~0.3-0.4 mm) of its
original diameter (0.8-1.2 mm).

6. Centrifuged for 5 min at 100 g .

7. Remove supernatant (debris) with a sterile Pasteur pipet carefully without disturb-
ing precipitated cells.

8. Add 5 mL of DMEM culture medium and pipet up and down several times
successively with smaller bore pipet tips (fi rst with a 1-mL tip, then with a
200- µ L tip) to resuspend cells.

9. Once thoroughly resuspended, cell concentration is counted on a Bright-line
hemocytometer with trypan blue (shake or invert well before use). Combine 20 µ L

of cell suspension with 30 µ L of DMEM culture medium and 50 µ L of 0.4%

L to hemocytometer.
Count on one central square (area = 1 mm 2 ; with 16 smaller squares) and each
of four 1-mm 2 squares located diagonally from the central square. Multiply the
total number of cells from the fi ve squares by 10,000 (if depth = 0.1 mm) to give
the concentration in the number of cells per milliliter. On an average, approx
600,000-1,000,000 cells can be yielded per neonate (from bilateral striata).
10. When counting cells, one can also determine the number of viable cells by dye
exclusion. Trypan blue is visible when it leaks into cells that have damaged
plasma membranes. By counting total cells and stained cells (damage cells) one
can calculate the percent viability ( see Note 4 ). In our practice, the cellular death
was usually <10% of total cells (2) .
11. Dilute with DMEM medium to a final concentration of 300,000 cells/mL.
Add 0.4 mL final suspension to each chamber of an eight-chamber glass

trypan blue (1 5 dilution of original suspension). Add 20 µ