User:David Benjamin Nyer/Notebook/PcTF breast cancer/2015/10/21

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Summary

Measured transfection efficiency of BT-549 using lipofectamine protocol, via fluorescence microscopy and flow cytometry. Measured transfection efficiency is approximately 45-50%.

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 1 mL PBS, then measured for FSC, SSC, and GRN fluorescence on the C6 flow cytometer.

Results

Fluorescence microscopy:

Well 4 - image 1

Well 4 - image 2

Well 5 - image 1

Well 5 - image 2

Well 6 (neg control)


Flow cytometry:

Well 4 - Count vs. GRN fluorescence
Image:2015-10-21_well_4_pMaxGFP.png

Well 5 - Count vs. GRN fluorescence
Image:2015-10-21_well_5_pMaxGFP.png

Well 6 (neg) - Count vs. GRN fluorescence
Image:2015-10-21_well_6_neg_ctrl.png

Conclusion & Next Steps

Transfection efficiency for pMaxGFP in BT-549 cell line is ~47%. Next steps are to perform optimization of Neon transfection system using BT-549 cell line.



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