Midbrain astrocyte and co-culture
gustavo.parfitt Parfitt
Abstract
Midbrain astrocyte and co-culture for mix-genetic experiments.
Steps
Intro
Midbrain NPC were generated following the publish protocol http://www.protocols.io/view/midbrain-organoid-differentiation-in-spinner-flask-rm7vzbnr4vx1/v1 with modifications listed bellow.
NPC generation
From a 80% confluent plate of iPSCs in a 10 cm plate.
Wash with 5ml of PBS
For the passaging add 3 mL of
Add 5 mL of StemFlex , gently mix and transfer to a 15 ml conical tube.
300rcf,25°C
Resuspend the cells in 1 mL of StemFlex with rock inhibitor and Count the cells using
Equipment
| Value | Label |
|---|---|
| Countess II | NAME |
| Life Technologies | BRAND |
| AMQAX1000 | SKU |
| http://www.thermofisher.com/us/en/home.html | LINK |
Plate 2 million cells in a 1/100
D0 change half the media with D0-1 medium.
D2 change half the media with D2-3 medium.
D3 Change half the media with D2-3 medium.
D4 change half the media with D4-7 medium.
D5 change half the media with D4-7 medium.
D6 change half the media with D4-7 medium.
D7 change half the media with D4-7 medium.
D8 change all the media with D8-11 medium.
D9 change half the media with D8-11 medium.
D10 change half the media with D8-11 medium.
D11 change half the media with D8-11 medium.
Freeze the NPCs in
NPC Plating
Thaw the NPCs and transfer to a 15 ml conical tube.
Add 5 ml of D12 terminal media with 10 μM Y-27632
300rcf,25°C
Resuspend the cells in 1 mL D12 terminal media with 10 μM Y-27632 and count the cells using
Equipment
| Value | Label |
|---|---|
| Countess II | NAME |
| Life Technologies | BRAND |
| AMQAX1000 | SKU |
| http://www.thermofisher.com/us/en/home.html | LINK |
Astrocyte co-culture
In a 96 well plate coated with
Change half media every day until day 25.
Obtain astrocytes following protocol http://www.protocols.io/view/astrocyte-extraction-from-brain-organoids-261ge364wl47/v2
Thaw the Astrocytes and transfer to a 15 ml conical tube.
Add 5 ml of A
300rcf,25°C
Resuspend the cells in 1 mL D12 terminal media with 10 μM Y-27632 and count the cells using
Equipment
| Value | Label |
|---|---|
| Countess II | NAME |
| Life Technologies | BRAND |
| AMQAX1000 | SKU |
| http://www.thermofisher.com/us/en/home.html | LINK |
Plate 10k on the neurons.
Mature the co-cultures until day 50 changing media every other day with day 25 media.
