Preparation of ventral midbrain cells for transplantation

Wash wells with 1 x PBS -/- 1. Incubate cells in 484µL of Accutase per cm² at 37°C. Note: D19 typically dissociate between 10-20mins.

1. Prepare a 15mL falcon tube for cells.
2. Check dissociation after 0h 6m 0s by using a p1000 to pipette up and down twice onto the cells. Observe if cells dislodge and are present in small clumps of 5-10 cells.
3. If YES: Collect all cells into a 15mL tube. Wash wells with Accutase to ensure all cells are collected.
4. If NO : Incubate plate for intervals of 0h 2m 0s Pipette suspension up and down twice targeting the large clumps/sheets at each interval to promote dissociation. Repeat until cells are dissociated into small clumps when you can transfer to a 15mL tube.

Add Ri (1:1000)1. Incubate the tube for 0h 1m 0s intervals until cells are broken into mostly single cells/small clumps (~5cells).

1. Cancel the reaction with PBS -/- at a 1:1 dilution to Accutase.
2. Spin cells (300x g,4°C)

Aspirate supernatant. Flick pellet twice.1. Resuspend in NBB27 + All (~2ml per 48well) + Ri 1:1000.

1. Resuspend suspension and pipette 10µL into a small Eppendorf tube for cell counting. Repeat for a second Eppendorf tube.
2. Take 2 tubes and add 10µL trypan blue to cells in each tube.
3. Transfer 10µL of mixed cells in haemocytometer.
4. Count cells in each quadrant.
5. Calculate total number of cells. Repeat this for tube 2 to ensure accurate cell counts.

Total cells = Average count of quadrants x Dilution factor x Volume (ml) x 10^4

8. Calculate the total volume needed to resuspend cells to a final density (typically between 100-150K/µl)and the amount of Ri 1:1000 required.

9. Spin cells (300x g,4°C)

Aspirate supernatant.1. Add half the required media on top of the cells gently (i.e. if you have 2 million cells total final volume is 20µLto achieve 100 000 cells/µL so add 10µLof media (NBB27 + All + Ri 1:1000) to pellet.

1. Using a P20 gently disturb the pellet in a circular motion, taking care not to damage cells by hitting the edge of the tube with the pipette tip.
2. Once mixed, take up the suspension once or twice and transfer to a small Eppendorf tube.
3. Measure the volume of cell suspension.
4. Add the appropriate volume of NBB27 + All + Ri 1:1000 required to reach the final volume.
5. Place cells on ice and transfer to surgical room immediately for transplantation.