Neurosphere culture: Difference between revisions
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== Material == | |||
Papain Mix: | '''Papain Mix:''' | ||
* 30U/ml Papain (Sigma Cat. No: P3125) | * 30U/ml Papain (Sigma Cat. No: P3125) | ||
* 0.24mg/ml cysteine (Sigma Cat. No: C7477) | * 0.24mg/ml cysteine (Sigma Cat. No: C7477) | ||
* 40µg/ml Dnase I Type IV (Roche Cat. No: 104 159) | * 40µg/ml Dnase I Type IV (Roche Cat. No: 104 159) | ||
Ovomucoid Mix: | '''Ovomucoid Mix:''' | ||
* 45mg trypsin inhibitor (Sigma Cat. No: T6522) | * 45mg trypsin inhibitor (Sigma Cat. No: T6522) | ||
* 21mg BSA (Sigma Cat. No: A3294) | * 21mg BSA (Sigma Cat. No: A3294) | ||
| Line 30: | Line 14: | ||
Sterile filtered!!! | Sterile filtered!!! | ||
Neurosphere Medium: | '''Neurosphere Medium:''' | ||
* DMEM:F12 + Glutamax (Gibco Cat. No: 31331-028) | * DMEM:F12 + Glutamax (Gibco Cat. No: 31331-028) | ||
* PenStrep (1:100) | * PenStrep (1:100) | ||
| Line 36: | Line 20: | ||
* FGF2 (20ng/ml) | * FGF2 (20ng/ml) | ||
== Procedure == | |||
Dissect SC from 5cm animals (GFP) | |||
Place SC into L15 medium (Gibco Cat. No: 31415-029) | |||
Remove meninges and cut SC in smaller pieces | |||
Transfer SC into PAP mix: Ovo mix (1:1), total volume 300µl | |||
Incubate at RT for 1.0 h | |||
Add 1 volume of additional Ovo mix | |||
Mix and incubate at RT for 5 minutes | |||
Dissociate the cells with a 1ml tip | |||
Add the cell suspension to 9ml of DMEM:F12 medium (Gibco Cat. No: 31331-028) at RT | |||
Centrifuge 3min at 700rpm | |||
Remove supernatant | |||
Add 1ml of Neurosphere medium and dissociate cells with 1ml tip | |||
Add an additional 3ml of Neurosphere medium and culture in 25ml flask at 25°C | |||
5% CO2 | |||
Keep the cells for 7-10 days until Spheres are forming | |||
=== Neurosphere transplantation === | === Neurosphere transplantation === | ||
Latest revision as of 08:05, 24 June 2015
Material
Papain Mix:
- 30U/ml Papain (Sigma Cat. No: P3125)
- 0.24mg/ml cysteine (Sigma Cat. No: C7477)
- 40µg/ml Dnase I Type IV (Roche Cat. No: 104 159)
Ovomucoid Mix:
- 45mg trypsin inhibitor (Sigma Cat. No: T6522)
- 21mg BSA (Sigma Cat. No: A3294)
- 390µl Dnase I Type IV (Roche Cat. No: 104 159)
- 39ml L15 medium (Gibco Cat. No: 31415-029)
Sterile filtered!!!
Neurosphere Medium:
- DMEM:F12 + Glutamax (Gibco Cat. No: 31331-028)
- PenStrep (1:100)
- B27 Supplement (1:50) (Gibco Cat. No: 17504-044)
- FGF2 (20ng/ml)
Procedure
Dissect SC from 5cm animals (GFP) Place SC into L15 medium (Gibco Cat. No: 31415-029) Remove meninges and cut SC in smaller pieces Transfer SC into PAP mix: Ovo mix (1:1), total volume 300µl Incubate at RT for 1.0 h Add 1 volume of additional Ovo mix Mix and incubate at RT for 5 minutes Dissociate the cells with a 1ml tip Add the cell suspension to 9ml of DMEM:F12 medium (Gibco Cat. No: 31331-028) at RT Centrifuge 3min at 700rpm Remove supernatant Add 1ml of Neurosphere medium and dissociate cells with 1ml tip Add an additional 3ml of Neurosphere medium and culture in 25ml flask at 25°C 5% CO2
Keep the cells for 7-10 days until Spheres are forming
Neurosphere transplantation
WT (white) animals around 3-4cm GFP-Neurospheres
anesthetize animals in 0.01% Bencocain remove skin and muscle with a tungsten needle and dissect a piece of SC from the side (Myotom 5 behind cloaca) add a Neurosphere, wait 5min until wound closure put the animal back in water wait for 7days that the NS can integrate in the SC cut the tail around 500µm behind the integrated NS and let the NS regenerate into the SC (6d Regenerate)