Apr 15, 2025
Human Primary Astrocyte (hPA) CRISPRa reprogramming protocol
- 1Duke University
- Gersbach Lab
Protocol Citation: Samuel Reisman, Charles Gersbach 2025. Human Primary Astrocyte (hPA) CRISPRa reprogramming protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov12477gr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 09, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 126469
Funders Acknowledgements:
NIH
Grant ID: HG012053
Abstract
This protocol describes methods for CRISPRa reprogramming of human primary astrocytes.
Materials
| A | B | C | |
| AGM | unit | 500 mL | |
| AGM (Sciencell 1801) | mL | remove 60ml from bottle | |
| FBS | mL | 50 mL | |
| AGM supplement (Sciencell 1801 supp) | mL | 5 mL | |
| P/S (Sciencell 1801 supp) | mL | 5 mL |
| A | B | C | D | E | |
| NDM (Guo 2014) | unit | 100 mL | 250 mL | 500 mL | |
| DMEM/F12 (GIBCO) | mL | 97.5 | 243.75 | 487.5 | |
| 0.5% FBS (GIBCO) | mL | 0.5 | 1.25 | 2.5 | |
| 3.5 mM glucose (Sigma)* | mg | 63.056 | 157.64 | 315.28 | |
| penicillin/streptomycin (GIBCO) | mL | 1 | 2.5 | 5 | |
| N2 supplement (GIBCO) | mL | 1 | 2.5 | 5 | |
| Filter sterilize after combining |
Supplements
| A | B | C | |
| Day | Action | Notes | |
| -1 | Coat plates with PDL | PDL is stored at -20 at 100x in 400ul aliquots (dilute to 40ml with sterile H2O) | |
| 0 | Prepare plates and transduce cells | ||
| 1 | Change media (AGM) | ||
| 2 | Change media (AGM + 1ug/ml Puro) | ||
| 3 | Change media (NDM + 1ug/ml Puro) | ||
| 4 | Check cells, refresh media if there is lots of debris | I usually change media here | |
| 5 | Check cells, refresh media if there is lots of debris | I usually change media here | |
| 6 | Change media to NDM without puro | ||
| 7 | Check cells, refresh media if there is lots of debris | I usually do not change media here | |
| 8 | Change media to NDM + BDNF (20ng/ml) + NT-3 (10ng/ml) | BDNF and NT3 stocks are both 1000x and are stored at -80C in 50ul aliquots | |
| 9 | Check cells, refresh media if there is lots of debris | I usually do not change media here | |
| 10 | Harvest. Proceed to sorting or RNA extraction | ||
| Be very gentle with all media changes and rinses |