User:David Benjamin Nyer/Notebook/PcTF breast cancer/2015/10/27

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Summary

Measured transfection efficiency of BT-474 using lipofectamine protocol, via fluorescence microscopy and flow cytometry. Measured transfection efficiency is approximately 11%.

Methods

See yesterday's page for transfection protocol. Cells were imaged using the Nikon TE inverted microscope at 100x magnification, using the 10x objective with phase contrast and GFP filter. Next, cells were washed with phosphate buffered saline, trypsinized and resuspended in 0.5 mL PBS, then measured for FSC, SSC, and GRN fluorescence on the C6 flow cytometer.

Results

Fluorescence microscopy:

Well 1 - image 1
2015-10-27 Well 1 phase GFP 10x 1.jpg

Well 1 - image 2
2015-10-27 Well 1 phase GFP 10x.jpg

Well 2 - image 1
2015-10-27 Well 2 phase GFP 10x 1.jpg

Well 2 - image 2
2015-10-27 Well 2 phase GFP 10x.jpg

Well 3 (neg control)
2015-10-27 Well 3 neg control phase GFP 10x.jpg


Flow cytometry:

Well 1 - Count vs. GRN fluorescence
2015-10-27 well 1 pMaxGFP.png

Well 2 - Count vs. GRN fluorescence
2015-10-27 well 2 pMaxGFP.png

Well 3 (neg) - Count vs. GRN fluorescence
2015-10-27 well 3 neg ctrl.png

Conclusion & Next Steps

Transfection efficiency for pMaxGFP in BT-474 cell line is ~11%. Next steps are to perform optimization of Neon transfection system using BT-474 cell line.