Author: NIH Stem Cell Lab
This is an extract of the Amaxa Biosystems protocol Vs. 09-2005 optimized for use with the UC06 cell line. It is suggested that you try all 5 programs (A-23, A-27, A-13, A-12, B-16) with each Nucleofection solution initially to determine the optimal program and solution for your specific hES strain.
Preparation of samples
- Collect hESC as for normal culture protocol using collagenase. Wash the cells with PBS after harvest, add Cell Dissociation Buffer (Gibco), and triturate to generate small clumps of approximately 50 cells (do not overtriturate). Wash again with PBS.
- Prepare 2ug circular DNA for transient expression or 10ug linearized DNA for stable clone selection for each sample in a volume ≤5 μl.
- Pre-warm the supplemented Cell Line Solution V to room temperature.
- Pre-warm one 500 μl aliquot of growth medium per sample (in a sterile microfuge tube) at 37°C.
- Prepare 6-well plates with 2 ml culture medium and pre-incubate the plates in a humidified 37°C/5% CO2 incubator. Plates should be plated with 4x10e5 MEF cells/well antibiotic-resistant feeder cells.
- Count the cells and determine cell density (cells/ml). Place the appropriate number of cells in a centrifuge tube, and centrifuge the cells at 100 x g for 10 minutes. Use 2x10e6 cells for transient experiments and up to 5x10e6 cells for stable expression.
- Resuspend the prepared cells in room temperature Nucleofector Cell Line Solution V to a final concentration of 1-2x10e6 cells/100 μl. Perform each sample separately to avoid storing the cells longer than 15 min in Nucleofector Solution.
- Mix 100 μl of cell suspension with the appropriate amount of DNA as described above.
- Transfer the sample into an amaxa-certified cuvette.
- Select the appropriate Nucleofector program—A-27 or B-16. (A-27 gives better survival, and B-16 gives better transfer efficiency.)
- To avoid damage to the cells, use 500 μl of media to remove the sample from the cuvette immediately after the program has finished. Gently transfer 500 μl of the pre-warmed recovery media from the microfuge tube to the cuvette. Gently remove the cell suspension from the cuvette, carefully return it to the microfuge tube, and place it in the 37°C incubator. It is critical to be gentle with the cells at this point, as they can be easily damaged by the shear stresses generated by pipetting. Do not mix the cell suspension by repeated pipetting.
Cultivation after nucleofection
- After 5 minutes, transfer the samples to the prepared 6-well plates. Transient gene expression can be observed within 12–18 hours after nucleofection.
- Wait at least 48 hours before selection for stable or homologous recombination transfectants.