DNA Transfection Into Stem Cells and Their Derivatives

Exceptional Transfection Efficiency in Human Induced Pluripotent Stem Cells (iPSCs) via Reverse Transfection with TransIT®-LT1. The TransIT®-LT1 Transfection Reagent was used to reverse transfect 1.3 x 10⁶ iPS cells with a ZsGreen expressing plasmid (Clontech). Reverse transfections were performed in 6-well plates using 12 µl of TransIT®-LT1 Transfection Reagent to deliver 4 µg of DNA (3:1, reagent: DNA). Cells were visualized 48 hours post-transfection and imaged under a 10X objective with an Olympus IX71® Inverted Microscope. Images are (A) phase contrast and (B) green fluorescence. Cells were assayed 48 hours post-transfection on an Accuri® Cytometer. The histogram (C) shows untransfected cells (black line) compared to cells transfected with plasmid using TransIT®-LT1 (green line). See more information on stem cell applications.

High Efficiency of Gene Expression in Mouse Embryonic Stem Cells (mESCs) using TransIT®-LT1. Mouse Embryonic Stem Cells (mESCs) were seeded at 250,000 cells per well of a 6-well plate and transfected two hours after plating with 6 μl of TransIT®-LT1 Transfection Reagent and 2.5 μg of a GFP expressing plasmid. Efficiency of transfection was visualized at approximately 60%. Images were taken using a Leica DMI 6000B inverted microscope 48 hours post-transfection. Images are (A) phase contrast, (B) green fluorescence, and (C) merged field.

Plasmid DNA Delivery to iCell® Cardiomyocytes Using TransIT®-LT1 Transfection Reagent. (A) High efficiency transfection of iCell® Cardiomyocytes with a GFP encoding plasmid. iCell® Cardiomyocytes were plated at 20,000 cells/well in a 96 well tissue culture plate coated with 0.1% gelatin. After allowing the cells to recover from thaw, cells were transfected with 100 ng/well of pMAXGFP (Lonza) using TransIT®-LT1 Transfection Reagent with a 2:1 reagent-to-DNA ratio according to the manufacturer’s instructions. Fluorescent images were taken 3 days post transfection using a Olympus IX71® inverted microscope. (B) cAmp induction measured via a luciferase reporter plasmid. iCell® Cardiomyocytes were plated for 5 days and subsequently replated using 40,000 or 80,000 cells/well in a 96 well plate pre-coated with gelatin. Three days post-replating cells were transfected using TransIT®-LT1 and a CRE-luciferase reporter plasmid. After 18 hours the cAMP pathway was induced using 10 µM isoproterenol for 6 hours. Luciferase activity was measured using the Promega Dual Glo® Luciferase Assay. Data is normalized to the control reporter.

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