Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/29

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RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines Main project page
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Summary

I'm going to make one more attempt at transiently transfecting U2OS cells with PcTF, then giving up on that and switching to a doxycycline-inducible, stable U2OS cell line. My hunch is that because U2OS cells grow so rapidly, the initial concentration of cells at the time of transfection is too high. To test this, I'll set up a 24-well plate with U2OS cells at low concentration, and take daily samples, in triplicate, to measure cell density using the flow cytometer.

I'm also preparing SKNSH cell line for transfection, as well as thawing the dox-inducible U2OS cells in preparation for passaging and eventual harvesting for RNA-seq.

Goals

  • Prepare more U2OS medium (McCoy's 5A w/ 10% FBS and P/S antibiotics)
  • Passage SKNSH cell line
  • Split SKNSH cell line into 6-well plate in preparation for transfection this week
  • Passage U2OS cell line
  • Split U2OS cell line into 6-well plate in preparation for transfection
    • Use very dilute cell concentration to prevent overgrowth of plate
  • Split U2OS cell line into 24-well plate to establish growth curve
    • Pull from same dilution that's used to seed 6-well plate
    • Be very precise when measuring out cell amounts to ensure good growth curve
  • Measure initial U2OS cell concentration using flow cytometry
  • thaw frozen U2OS stock w/ doxycycline-inducible PcTF gene, begin PS-α

Protocol

Mammalian Culture Media
Thawing Cells
Splitting Cells
Flow cytometry for mammalian cells

Materials

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

Notes

For the 6-well plate containing SKNSH cell line:

  • total volume = 4mL * (6 + 1 extra well) = 28 mL
  • Approx. 10E6 cells per T-75, or 1E6/mL
  • Desired final concentration of 2E5 cells/well, or 0.2mL of T-75 stock
  • 0.2*(6+1) = 1.4mL stock
  • in a 50mL conical, add 1.4mL of stock + 26.6mL of medium = 28mL total
  • aliquot 4mL diluted cells into each well, with 4mL left over

For the 6-well and 24-well plates containing U2OS cell line:

  • After trypsinizing the cells, adding 8mL medium to stop trypsinization, dilute 4-fold in a 15mL conical and use as stock
  • total volume = 4mL * 6 + 1mL * 24 = 48mL, round up to 50mL for safety
  • 0.2*6 + 0.05*(24+ 2 extra) = 2.5mL stock
  • in a 50mL conical, add 47.5mL medium + 2.5mL stock = 50mL total
  • aliquot 4mL diluted cells into each 6-well and 1mL diluted cells into each 24-well.
  • Use a 1mL pipettor for this, as we want to have exact volumes
  • Keep the remainder for flow cytometry

NOTE: Only 250uL of diluted cell suspension remained after portioning out to 6 and 24 well plates. Make calculations accordingly.

Results/Conclusions

Measured cell density of U2OS cells using flow cytometry: 5 cells/uL

This is diluted 4x (because 250uL culture was replaced with 1mL FC buffer), so actually 20 cells/uL

20,000 cells/mL initial concentration



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