Apr 20, 2026

Lentiviral vector production and primary astrocyte transduction

 Forked from Lentivirus Production and Astrocyte Transduction
DOI
https://dx.doi.org/10.17504/protocols.io.x54v9rkb1v3e/v1
  • 1Duke University;
  • 2KU Leuven;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v9rkb1v3e/v1
Protocol Citation: Shiyi Wang, Sarah van Veen 2026. Lentiviral vector production and primary astrocyte transduction. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9rkb1v3e/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: January 08, 2025
Last Modified: April 20, 2026
Protocol  Integer ID: 117922
Keywords: ASAPCRN, lentivirus, lentiviral transduction, primary astrocytes, lentiviral vector production, generating lentivirus, production of lentivirus, transduction into astrocyte, transducing astrocyte, primary astrocyte transduction, primary astrocyte transduction this protocol, lentivirus, primary astrocyte, astrocyte, primary gene function, study of primary gene function
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
This protocol describes the production of lentivirus and their transduction into astrocytes. It provides detailed steps for generating lentivirus, transducing astrocytes efficiently, and selecting for stable transduction, enabling the study of primary gene function in primary astrocytes.
Materials
  • pLKO.1 shRNA Puro targeting plasmid (for astrocyte transduction)
  • Envelope plasmid (VSVG)
  • Packaging plasmid (dR8.91)
  • X-tremeGENE HP DNA Transfection Reagent (Roche, Cat# 6366236001)

Astrocyte growth media (AGM)
  • DMEM (GIBCO, Cat# 11960)
  • 10% FBS (Thermo Fisher, Cat# 10-437-028)
  • 10 μM hydrocortisone (Sigma, Cat# H0888)
  • 100 U mL−1 Pen/Strep (GIBCO, Cat# 15140)
  • 2 mM L-glutamine (GIBCO, Cat# 25030-081)
  • 5 μg mL−1 insulin (Sigma, Cat# I1882)
  • 1 mM Na pyruvate (GIBCO, Cat# 11360-070)
  • 5 μg mL−1 N-acetyl-L-cysteine (Sigma, Cat# A8199)) 

Safety warnings
  • Handle lentiviruses in a biosafety level 2 (BSL-2) laboratory following institutional guidelines.
  • Ensure proper disposal of materials used for lentivirus production according to institutional biohazard waste disposal protocols.
  • Perform all steps involving lentivirus production under sterile conditions to prevent contamination.
Plate HEK293T cells (day -1)
15m
Plate HEK293T cells in appropriate culture vessels (e.g., T75 flask) to achieve 70-80% confluency on the day of transfection.
Transfection of HEK293T cells with lentiviral plasmids (day 0)
15m
Prepare DNA tubes:
  • 10.5 ug of your plasmid
  • 3.5 ug of VSVG plasmid
  • 7.0 ug of dR8.91 plasmid
  • 1 ml OptiMEM
Add 60 ul of X-tremeGENE to 1ml of OptiMEM, for each flask.
Mix DNA solution and X-tremeGENE solution and incubate at room temp for 00:15:00 .

15m
Use a 5ml pipet to add transfection mixture dropwise to flask and gently swirl flask to mix.
Incubate Overnight at 37°C with 5% CO2.

Replace media (day 1)
Aspirate media from flask.
Replace with 12 ml of Astrocyte Growth Media (AGM).
Collect lentivirus (day 2)
Collect media from flask in a 15 ml tube.
Add 12 ml of AGM to flask and put back in the incubator.
Spin down collected media for 10 min at 1100 rpm to pellet any dead cells/debris.
Filter collected media through a 0.45 μm syringe filter.
Test for presence of viral particles using Lenti-X GoStick (Clonetech). Add 20 ul of media to the stick and then add 4 drops of chase solution.  The control band will appear right away, and within 5-10 min you should see a faint second band appear if there is virus present.
Aliquot lentivirus and store at -80°C for future use.
Collect lentivirus and discard cells (day 3)
Collect media from flask in a 15 ml tube.
Add bleach solution to flask to kill remaining viral particles, aspirate, and discard flask.
Spin down, filter and aliquot media as described in previous section.  Don’t need to test with a Lenti-X GoStick this time.

Transduction of rat primary astrocytes
Plate rat primary astrocytes (at DIV7) in 6-well dishes at a density suitable for transduction experiments (300,000-400,000 cells/well).

For details on the astrocyte isolation protocol, refer to the protocol below.
Protocol
NAME
Astrocyte Isolation and ACM Production
CREATED BY
Justin T Savage


Get a bucket of ice and get lentivirus aliquot from -80°C.
Remove 500 ul of media from each well you wish to treat.
Add 500 ul of lentivirus, and 1ug/mL polybrene.
Note
Optimize lentiviral titration to achieve desired transduction efficiency in astrocyte cultures.

Two days after addition of lentivirus, begin puromycin treatment (astrocytes are DIV10).
Add some AGM to a 50 ml tube and add puromycin at 2ug/mL. Equilibrate media.
Remove 1 ml of media from astrocytes, add 1 ml of media + puromycin. Final puromycin concentration is 1ug/mL. 
After two days (astrocytes are DIV12), repeat puromycin feeding by feeding with AGM + puromycin at 2ug/mL.
After three days (astrocytes are DIV15, day 5 of puromycin treatment), lyse astrocytes.
Protocol references
Christabel Xin Tan, Dhanesh Sivadasan Bindu, Evelyn J. Hardin, Kristina Sakers, Ryan Baumert, Juan J. Ramirez, Justin T. Savage, Cagla Eroglu; δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte–neuron cadherin interactions. J Cell Biol 6 November 2023; 222 (11): e202303138. doi: https://doi.org/10.1083/jcb.202303138

Katherine T. Baldwin, Christabel X. Tan, Samuel T. Strader, Changyu Jiang, Justin T. Savage, Xabier Elorza-Vidal, Ximena Contreras, Thomas Rülicke, Simon Hippenmeyer, Raúl Estévez, Ru-Rong Ji, Cagla Eroglu,
HepaCAM controls astrocyte self-organization and coupling, Neuron, Volume 109, Issue 15, 2021, Pages 2427-2442.e10, ISSN 0896-6273, https://doi.org/10.1016/j.neuron.2021.05.025.

Stogsdill, J., Ramirez, J., Liu, D. et al. Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis. Nature 551, 192–197 (2017). https://doi.org/10.1038/nature24638