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

PcTF breast cancer

Main project page Previous entry      Next entry

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 Well 5 - Count vs. GRN fluorescence Well 6 (neg) - Count vs. GRN fluorescence 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.