Hi!
I recently tried to transfect WTC10 stem cells with a gene under the EF1a promoter. ~30k cells were seeded per well of a CellVisC4-1.5H-N chambered slide coated with vitronectin, in mTeSR+ medium with 1X RevitaCell and P/S. ~24h post plating, media was changed to fresh mTeSR+ media (with RevitaCell and P/S), and transfection was performed with 500 ng DNA and 1/2 uL Lipofectamine STEM in OptiMEM medium (50 uL/well). 24 h post transfection, media was changed to mTeSR+ (no RevitaCell, P/S), and imaging was performed ~40h post transfection. I observed a really low transfection efficiency (maybe 1 in 100 cells), and was wondering if you had any tips/protocol suggestions to improve this. The cells already had 2 genes endogenously tagged.
Thanks!
Hi and thanks for your question! We don’t use lipofection very often (it’s not a part of our routine cell line generation), but I’m happy to share the general steps in our internal protocol. We work with WTC11 hiPSCs, use Matrigel as our substrate, and feed with mTesR1 medium. We typically do lipofections in 96wp format. We seed ~5k cells/well, use ~50-100ng DNA/well, and use 0.2-0.4uL LipoStem/well (diluted in OptiMem) as recommended by the LipoStem protocol. We include Rho kinase inhibitor when seeding cells for transfection, and feed them fresh media (including Rho kinase inhibitor) the day of transfection with preference for feeding them a least an hour before transfection will take place. Using this method, for plasmids encoding fluorophores driven by strong/constitutive promoters (like CAGs, prepped endotoxin-free), we’ve been able to accomplish >50% efficiency with high frequency. We typically observed more cell death for higher DNA/Lipo concentrations, but cell death in general is relatively minimal.
Given your seeding density you could try using more Lipofectamine to match Thermo’s recommendations for LipoStem in 24wp format (1-2uL I believe), but I’m not sure your lower dosage of LipoStem is the sole culprit for such a low efficiency. Your protocol scheme in general looks reasonable, so I would double-check things like DNA purity/quantity, LipoStem expiration, etc - the little stuff. Hope that helps!
