Nov 28, 2022

Public workspaceiPSC Transduction

DOI
dx.doi.org/10.17504/protocols.io.q26g7y1y1gwz/v1
  • 1Massachusetts General Hospital;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Daniel Choo
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DOI: dx.doi.org/10.17504/protocols.io.q26g7y1y1gwz/v1
Protocol Citation: Ali Albalakhi, Ning Xia 2022. iPSC Transduction. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y1y1gwz/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: November 28, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 73279
Keywords: ASAPCRN
Abstract
This is the iPSC transduction protocol.
Attachments
Icon for Transduction Protocol .docx
Transduction Protoco...
38KB
Guidelines
Note:
Cells will need to be maintained in StemFlex medium to reform healthy iPSCs colony-like morphology (4-5 days)
Given that we don’t know what viral titer will be toxic to the cells during transduction we will have to optimize the transduction as we progress
Materials
Material needed:
  1. Packaged virus
  2. Polybrene (diluted 100µg/mL)
  3. Stem flex (+) warmed to room temperature ~ 10 to 15min
  4. Vitronectin-coated plates (coating to final concentration of 1.0µg/cm2)
Begin transduction with cell line 7026
Begin transduction with cell line 7026

Day 1:
Day 1:
Split iPS cells into single cells in complete medium with RVC. (500µl per well)
Seed cells at 5.0X104 cells in glass-bottom 24-well plates
Day 2: Calculations are for 24-well plates. (If using a 6 well plate, then seed at 1x105 cells per well, 850µL of medium the next day with 150µL)
Day 2: Calculations are for 24-well plates. (If using a 6 well plate, then seed at 1x105 cells per well, 850µL of medium the next day with 150µL)
Virus infection:
Remove the culture medium from cells, add (300µL) fresh medium without RVC into each well

Add 45µl virus and 1µg/ml polybrene into cells and mix it. Incubate in 37 degree, 5% CO2.



Well 1Well 2Well 3Control
PLVX mCherry HLA-A2PLVX IRES-Puro HLA-A2 pLoc Mlana


Add Polybrene into the infected well to make the final concentration to be 1ug/ml.
Stock Polybrene (10mg/mL)
Make a 100x dilution to make a working concentration of 100µg/mL

(10mg/mL) (Vstock) = (100µg/mL)(0.5mL)
Vstock = 5µL of stock into 495µL of DPBS (-)(-)

(100µg/mL) (Vstock) = (1µg/mL)(0.3mL) --> Vstock = 3µLof working concentration per 300µL of medium added
Day 3:
Day 3:
Monitor the cells for the next 48 hours
Day 4 (or 48 hours later):
Day 4 (or 48 hours later):
Check IRES-GFP or HLA-A2’s expression at least 48 hours after the transduction.
For antibiotic-based selection, the cells will be maintained to reform colonies and subsequently grown in medium with 1µg/mLof Puromycin to initiate selection