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

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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:
2015-06-01 Well 1 pMaxGFP Method 1 phase GFP 10x.jpg

Well 2:
2015-06-01 Well 2 pMaxGFP Method 2 phase GFP 10x.jpg

Well 3:
2015-06-01 Well 3 pMaxGFP Method 3 phase GFP 10x.jpg

Well 4:
2015-06-01 Well 4 KAH126-MV2 Method 1 phase mCherry10x.jpg

Well 5:
2015-06-01 Well 5 KAH126-MV2 Method 2 phase mCherry10x.jpg

Well 6:
2015-06-01 Well 6 KAH126-MV2 Method 3 phase mCherry10x.jpg

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.