Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/31

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Owwnotebook icon.pngRNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines Report.pngMain project page
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Summary

Repeat of yesterday's work.

Checking on the U2OS & SKNSH cell lines today. I'll take three wells from the 24-well plate and 1 well from the 6-well plate, trypsinize the cells to get them into solution, then measure their density on the flow cytometer. Should be able to get a rough idea of when the plates will be ready for transfection then.

I'll also be taking pictures of the plates in order to correlate measured cell density (via FC) with estimated confluence.

Goals

  • Use flow cytometry to measure cell density of U2OS cultures in wells
  • Take photographs of U2OS & SKNSH cells in wells

Protocol

flow cytometry for mammalian cells

Materials

  • List materials here, to be gathered before the experiment
  • Consumables such as media, antibiotics, DNA, kits etc.

Notes

Observations, measurements, placeholder info etc. go here

Results/Conclusions

Flow cytometry data:

Plot 1 (FSC-A/SSC-A)
All P2
Count Volume (µL) Events / µL Count Events / µL % of All
A01 Blank 820 100 8 1 0 0.12%
A02 Well 2 (6-well plate) 18,045 100 180 15,098 151 83.67%
A03 Well A1 (24-well plate) 3,517 100 35 2,552 26 72.56%
A04 Well B2 (24-well plate) 3,967 100 40 2,734 27 68.92%
A05 Well C3 (24-well plate) 2,935 100 29 1,414 14 48.18%


U2OS culture confluency on the right:

U2OS

It appears cell density is somewhere between 150,000 cells (in 4mL of medium) and 100,000 cells (in 1 mL of medium, corrected for volume). Will perform transfection tomorrow.