Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/06/01

K562 Neon Transfection Results, Day 5

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Overview After transfection on Wednesday the 27th, the K562 cells were expanded on Friday the 29th from 1mL cultures in 12-well plates to 4 mL cultures in 6-well plates. Today I collected more images of the cells using fluorescence microscopy and measured the percentage of fluorescent cells in culture using flow cytometry. Fluorescence Microscopy Well 1: Well 2: Well 3: Well 4: Well 5: Well 6: Observations: Lots of cell debris/dead cells in wells 3, 5, and 6. Looks as though method #3 (wells 3 & 6) is particularly hard on the cells. Dead cells have smaller diameter, more complex internal structures compared to live cells. Flow Cytometry Plot 1 (FSC-A/SSC-A) Plot 3 (FL1-A [GRN]) Plot 4 (FL3-A [RED]) All P2 M2 M1 Count Volume (µL) Events / µL Count Events / µL % of All Count % of This Plot Count % of This Plot Blank 813 20 41 0 0 0.00% 0 100.00% 0 100.00% Untreated 11,498 10 1150 9,474 947 82.40% 1 0.01% 6 0.06% GFP 1 19,682 10 1968 16,646 1665 84.57% 7,836 47.07% 297 1.78% GFP 2 12,256 10 1226 9,637 964 78.63% 8,025 83.27% 902 9.36% GFP 3 904 10 90 20 2 2.21% 17 85.00% 8 40.00% KAH126 1 7,648 20 382 3,615 181 47.27% 10 0.28% 32 0.89% KAH126 2 3,850 20 193 592 30 15.38% 5 0.84% 30 5.07% KAH126 3 3,034 20 152 332 17 10.94% 4 1.20% 20 6.02% Untreated 12,735 10 1274 10,205 1021 80.13% 1 0.01% 3 0.03% Blank 1,069 20 53 97 5 9.07% 0 0.00% 0 0.00% Transfection efficiency appears low (<5%) for all of the KAH126 samples. Will consult with Dr. Haynes and see about repeating the experiment using higher cell density and higher concentration of plasmid.