Dec 12, 2025
Transduction of Microglia-like cells (iMG) with lentivirus
- Elisa Perciballi1,
- Saša ereb2,
- Martine Therrien1,
- Beth Stevens3
- 1University of California, Davis;
- 2Broad Institute;
- 3HHMI, Broad Institute, Boston Children's hospital
Protocol Citation: Elisa Perciballi, Saša ereb, Martine Therrien, Beth Stevens 2025. Transduction of Microglia-like cells (iMG) with lentivirus. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrw1xolmk/v1
Manuscript citation:
Dolan, M.-J., Therrien, M., Jereb, S., Kamath, T., Gazestani, V., Atkeson, T., Marsh, S.E., Goeva, A., Lojek, N.M., Murphy, S., et al. (2023). Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro. Nat. Immunol. 24, 1382–1390. https://doi.org/10.1038/s41590-023-01558-2.
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: December 08, 2025
Last Modified: December 12, 2025
Protocol Integer ID: 234434
Keywords: stem cell, microglia, lentivirus, transduction with lentivirus, lentiviral rna, reverse transcription of lentiviral rna, lentivirus ipsc, transduction of microglia, derived microglia, microglia protocol, lentivirus, encoded protein vpx, protein vpx, derioved microglia protocol, microglia, role of microglia, immune alteration, immune alterations during development, dna delivery, vpx, resistant to dna delivery, virus
Funders Acknowledgements:
Alzheimer’sAssociation
Stanley Center for Psychiatric Research
Cure Alzheimer’s Fund
Howard Hughes Medical Institute
Abstract
iPSC-derived microglia are an important tool for studying the role of microglia and neuro-immune alterations during development, adulthood, and aging, both in health and disease. However, microglia are resistant to DNA delivery, so following this protocol, transduction with lentiviruses is facilitated by the SIV-encoded protein Vpx, which degrades SAMHD1, a restriction factor that prevents reverse transcription of lentiviral RNA. Co-delivery of Vpx packaged in virus-like particles (VLPs) leads to ~89% transduction in iMGs, compared with ~4% of iMGLs with lentivirus alone (Dolan, Therrien et al 2023). This protocol is used in combination with the iPSC-derioved microglia protocol (10.17504/protocols.io.ewov11rw2vr2/v1)
Protocol materials
TransIT®-LT1 Transfection ReagentMirus BioCatalog #MIR 2304
Opti-MEMLife TechnologiesCatalog #31985070
Lenti-X ConcentratorTakara Bio Inc.Catalog #631232
DMEM-F12 medium Gibco - Thermo Fisher ScientificCatalog #31331093
Troubleshooting
Before starting
This protocol is designed for transduction of microglia at Day 35 of differentiation. Because of this stress, transduced cells need stabilization in the maturation process; therefore, it is recommended to perform desired experiments at final time-point Day 42 instead of Day 40.
| A | B | |
| Vpx (plasmid #132928) | Addgene; https://www.addgene.org/132928/ | |
| VSV-G (plasmid #8454) | Addgene, https://www.addgene.org/8454/ | |
| psPAX2 (plasmid #12260) | Addgene; https://www.addgene.org/12260/ |
Plasmids needed
Procedure for lentivirus production in HEK293T cells:Untitled section
5d 13h 20m
Day 1: Seed 6 x 105 HEK293T cells/well in 6-well plates in DMEM with 10% FBS without antibiotics.
Note
Number of cells at plating might need to be adjusted depending on cell growth
Day 2: Transfection
- Warm the TransIT-293 reagent at room temperature and vortex gently before use.
- Place 250 μl of Opti-MEMLife TechnologiesCatalog #31985070 per well in a sterile tube.
- Add 3 μg of plasmid DNA per well to the respective tube (see tables below for details).
- Pipet gently to mix all the components completely.
- Add 8 µL of TransIT®-LT1 Transfection ReagentMirus BioCatalog #MIR 2304 Reagent to the diluted DNA mixture and pipet gently again.
- Incubate at room temperature for 00:30:00 .
- Distribute the complexes to cells in complete growth medium drop-wise, rock the plate and transfer in normoxic incubator (20% O2, 5% CO2, 37 °C ).
| A | B | |
| Component | μg of plasmid DNA/well | |
| Vpx (pSIV3) plasmid | 2.6 | |
| VSV-G plasmid | 0.4 |
For producing viral-like particles loaded with Vpx protein in 6-well plate (for 1 well containing 2 mL media)
| A | B | |
| Component | μg of plasmid DNA/well | |
| Transfer plasmid | 1.6 | |
| VSV-G plasmid | 0.4 | |
| psPAX2 plasmid | 1 |
For producing target gene lentivirus in 6-well plate (for 1 well containing 2 mL media)
Note
In order to have an efficient transfection, it is important to mix well the reagents both in the tube and in the well.
30m
Day 3: 18:00:00 after transfection, change media to 2 mL Opti-MEMLife TechnologiesCatalog #31985070
18h
Day 4: 24:00:00 to 72:00:00 after transfection collect the virus and concentrate
4d
Collect virus
- Harvest viral supernatant and collect in a 15 ml conical tube.
- Centrifuge viral supernatant at 300 x g, 00:05:00 to remove any HEK293T cell in the supernatant. Alternatively, filter through a 0.45 μm filter.
Note
Determine the best incubation time post-transfection for each virus type. The optimal
incubation time is generally 24–72 hours, but will vary depending on the goal of the
experiment, nature of the plasmid used, and cell doubling time.
5m
Concentrate the virus, if necessary
- Transfer supernatant to a new tube. Add 1/3 of the volume ofLenti-X ConcentratorTakara Bio Inc.Catalog #631232 to the viral supernatant and incubate at least 18:00:00 4 °C
Note
Concentration of the lentivirus is only required for viruses with large viral genomes. Viruses with small genomes are produced at higher titers and may not need to be concentrated.
Vpx particles do not need to be concentrated.
18h
Day 5: Finalize virus concentration
- Centrifuge the tubes at 1500 x g, 4°C, 00:45:00 to spin down the lentivirus with Lenti X concentrator.
- Resuspend the virus in 1/10th of the original volume with iMG medium or DMEM-F12 medium Gibco - Thermo Fisher ScientificCatalog #31331093 .
- VPX particles and lentiviruses can be stored frozen at -80 °C .
Note
Avoid cycles of thawing and refreezing, so aliquot desired amount of Vpx particles and viruses and only thaw prior using it.
45m
Lentivirus titration:
For efficient microglia transduction, the amount of virus is 53 ng of P24/10,000 cells. In order to determine the virus titer, follow the Lenti-X p24 Rapid Titer Kit User Manual.
Note
Vpx cannot be measured by p24 lentivirus assay. In our hands, usually, 10 µl of Vpx/10,000 cells allows robust infection of microglia.
iMG Transduction
1d 14h
Transduction is done on iMG at Day 35 of differentiation.
- If using a multi-well plate, adjust volume across the wells ( 96-well plate to 50ul) so it is equal across the plate. Keep the conditioned media to use on Day 36.
- Based on the virus titer obtained, calculate the volume needed to have 53 ng P24/10,000 cells + 10ul of Vpx.
- Combine Vpx particle and lentivirus , gently mixing doing up and down.
- Add appropriate volume to each well.
Day 36 of differentiation: 18:00:00 - 20:00:00 after infection
- Remove media in each well.
- Add 50 µL of the conditioned medium collected on Day 35 and 50 µL of Mg basal differentiation medium supplemented with Day 30 growth factors (refer to the tables below).
1d 14h
Day 38, 40: Feed iMG
- Add 50ul of fresh Mg basal differentiation medium supplemented with Day 30 growth factors
Day 42: Assess cells
Note
All experiments reported in Dolan, Therrien et al. (2023) were performed on day 42, 7 days after transduction. This time point allows iMG to recover after transduction and show normal morphology and transcriptional profile. Timing can vary depending on the experiments.