Passaging Neural Stem Cells
| Title | Passaging Neural Stem Cells |
| Date Submitted | May 5, 2012 |
| Submitted by - | Efthymiou, Anastasia - anastasia.efthymiou@nih.gov |
| Adapted from - | Gibco Protocol |
| Contributors - | Efthymiou, Anastasia |
| Affiliation(s) - | NIH CRM - NIAMS – Laboratory of Stem Cell Biology |
Introduction:
Protocol:
1. Aspirate the medium and wash with D-PBS without Ca2+ and Mg2+.
2. Add 1 mL of TrypLE Express or StemPro Accutase to the culture vessel.
Note: The monolayer lifts off from the culture dish within 30 seconds of application of TrypLE Express or StemPro Accutase.
3. Gently pipette to loosen monolayer into a single cell suspension. Neutralize the treatment by adding 4 mL of medium. Do not treat the cells for longer than 3 minutes after addition of TrypLE Express or StemPro Accutase.
4. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate and discard the supernatant.
5. Resuspend the cells in StemPro NSC SFM complete medium.
6. Count the cell number
7. Plate cells in fresh medium on a CELLstart CTS - or Fibronectin-coated plate at a density of 1 × 10^4 to 1 × 10^5 cells/cm2, or split the cells at a 1:4 ratio.
Materials:
Neurospheres
D-PBS without Ca++ and Mg++
TrypLE Express or StemPro Accutase
StemPro NSC SFM complete medium
CELLstart CTS or Fibronectin-coated plate
StemPro NSC SFM complete medium
| Component | Final concentration | Amount |
| KnockOutTM D-MEM/F-12 | 1X | 97 mL |
| GlutaMAXTM-I Supplement | 2 mM | 01 mL |
| bFGF | 20 ng/mL | 20 µL |
| EGF | 20 ng/mL | 20 µL |
| StemPro® Neural Supplement | 2% | 2 mL |
