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

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K562 Neon Transfection Results, Day 5 Main project page
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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])
AllP2M2M1
CountVolume (µL)Events / µLCountEvents / µL% of AllCount% of This PlotCount% of This Plot
Blank8132041000.00%0100.00%0100.00%
Untreated11,4981011509,47494782.40%10.01%60.06%
GFP 119,68210196816,646166584.57%7,83647.07%2971.78%
GFP 212,2561012269,63796478.63%8,02583.27%9029.36%
GFP 390410902022.21%1785.00%840.00%
KAH126 17,648203823,61518147.27%100.28%320.89%
KAH126 23,850201935923015.38%50.84%305.07%
KAH126 33,034201523321710.94%41.20%206.02%
Untreated12,73510127410,205102180.13%10.01%30.03%
Blank1,06920539759.07%00.00%00.00%

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.



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