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

RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines

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Summary Happy New Year! Let's do some science. The U2OS culture is at optimal density, so I'll be performing another transfection today. Also continuing to measure growth on the 24-well plate using flow cytometry, and advancing the KAH126/U2OS culture from PS-α to PS-β. Goals Transfect 3 U2OS cultures from 6-well plate with PcTF-RFP plasmid (and use 1 well as negative control) Take photographs of cultures to show confluency levels Measure cell density on 3 U2OS cultures from 24-well plate using flow cytometry Advance KAH126/U2OS cell line from PS-α to PS-β Protocol transfection advancing cells flow cytometry Materials PcTF-RFP plasmid (at room temp.) Lipofectamine kit (at room temp.) Opti-MEM (at room temp.) antibiotic-free complete U2OS medium (at 37°C) complete U2OS medium (at 37°C) FACS buffer (PBS and 1% FBS) at 4°C pipette tips, etc. Notes Observations, measurements, placeholder info etc. go here Results/Conclusions Photograph of the culture is on the right. Near-total confluency observed. Below is the table data from flow cytometry of the 24-well samples. 2015-01-01, phase 10x All P2 Count Volume (µL) Events / µL Count Events / µL % of All A01 Blank 635 100 6 0 0 0.00% A02 Well A6 7,381 100 74 7,381 74 100.00% A03 Well C1 7,734 100 77 7,734 77 100.00% A04 Well D2 6,814 100 68 6,814 68 100.00% Average cell density of 73,000 +/- 4600 cells/mL is observed. This translates to a cell count of approximately 292,000 cells per well in the 6-well plate when transfection occurred. KAH126/U2OS cell line was not advanced due to lack of T-75 culture flasks. Will try again tomorrow and see about borrowing flasks from another lab.