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

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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.