A: Currently, we can only recommend using this mix with fibroblasts.
A: Our protocol is optimized using Lipofectamine™ MessengerMax™, and we strongly recommend using it without substitution. Substituting another reagent may lead to loss of reprogramming efficiency.
A: Synthetic mRNAs produced without chemically modified nucleotides along with the double-stranded RNA (dsRNA) byproducts generated during in vitro transcription (IVT) can activate the innate immune response in cells. This activation may lead to a cascade of cellular events, including the release of inflammatory cytokines, suppression of protein synthesis, cell cycle arrest, and apoptosis. Based on our observations, a single transfection of synthetic mRNA typically does not compromise the overall viability of a cell culture. However, repeated transfections—such as those required during cellular reprogramming—can significantly amplify the innate immune response, resulting in extensive cell death and impairing the efficiency of reprogramming and the formation of induced pluripotent stem cell (iPSC) colonies.
The mRNAs in the INCOGNITO™ iPSCMax™ Ψ-mRNA Reprogramming Mix contain the modified nucleoside pseudouridine substituted for each uridine in the mRNA sequences and have been treated with CELLSCRIPT™’s Min-Immune™ Gold dsRNA Removal Kit, producing an mRNA mix with ultra-low immunogenicity that enables high reprogramming efficiencies.
A: No. Our reprogramming procedure using the INCOGNITO™ iPSCMax™ Ψ-mRNA Reprogramming Mix is performed using a standard cell culture incubator set at 37°C and 5% CO2.
A: For reliable reprogramming, use adult dermal or neonatal fibroblasts. Aim for low-passage, actively dividing cultures, as these respond far better to mRNA transfection and are easier to reprogram.
A: Yes. Early-passage fibroblasts reprogram more efficiently. With higher passage, fibroblasts may enter senescence, making them more sensitive to the stresses of mRNA transfection and harder to reprogram.
A: It’s possible but expect lower efficiency. Senescent fibroblasts are more sensitive to stresses induced by mRNA transfection and are harder to reprogram. If you must use them, you may need to extend the regimen (e.g., 7–10 transfections instead of 5) and/or lower the mRNA dose delivered with each transfection to mitigate the death that may occur due to stress induced by the mRNA transfections.
A: Look for cells that are larger, flatter, and slower to divide compared to compact, fast-dividing fibroblasts. Morphology and the rate at which a culture needs splitting (e.g. every 3-4 days versus every 5-7 days) are good first clues. In research settings, senescence-associated markers (e.g., SA-β-gal staining) can provide confirmation.
A: Start with the recommended baseline (e.g., 500 ng mRNA per transfection per well in a 6-well plate with the recommended transfection reagent volume and plated cell density). If efficiency is low, adjust systematically:
- Modify mRNA or transfection reagent amounts
- Alter cell density at plating
- Extend the number of transfections (up to 10 if needed)
A: Absolutely. While 500 ng mRNA per well is standard, you can fine-tune mRNA and reagent volumes depending on your fibroblast line’s viability and transfection sensitivity. However, we do NOT recommend going below 300 ng of the mRNA mix per transfection per well at the recommended cell density of 5×105 cells per well.
A: Expect colonies to emerge 3-5 days after the final transfection. Once colonies are large enough, manually pick them onto plates coated with an appropriate substrate (e.g. Laminin-521, Vitronectin, or Matrigel®) in iPSC medium supplemented with 10 µM Y-27632 ROCK inhibitor (only for the first 24 hours after transfer).
A: No. ROCK inhibitor is used only during the first 24 hours post-picking to improve survival. After that, maintain colonies in standard iPSC medium without the inhibitor.
