Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/15: Difference between revisions

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*'''[[User:David Benjamin Nyer|David Benjamin Nyer]] 18:18, 15 December 2014 (EST)''':
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Revision as of 16:18, 15 December 2014

RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Summary

After starting the SKNSH cell lines in small (T25) flasks, they need to be transferred to a larger container to increase biomass in preparation of experimentation. If the initial thawing and starting of a cell line is Passage Alpha, the first transfer into a T75 flask is Passage Beta. The cells will need to have their medium removed, their attachment to the flask disrupted with trypsin-EDTA, and then be transferred into fresh media.
After advancing the U2OS cell lines into medium (T-75) flasks, they need to be split into more containers to increase biomass, portion for experiments, restore cryo stocks, and maintain the cell line. If kept in the same container for too long, cells will enter senescence and no longer be competent for transfection. Therefore it's important to continue splitting cells and to experiment on cell lines that are still relatively young.
It's important to keep in mind that not all cell lines develop at the same rate. The U2OS line grows significantly faster than SKNSH, and will therefore require more frequent splitting/passaging.

Goals

  • Passage U2OS cells from PS-β to PS-1
  • Split U2OS cells into 6-well plate for experiment
  • Prepare stocks of U2OS cells for cryopreservation
  • Advance SKNSH cells from PS-α to PS-β

Protocol

advancing adherent cells
passaging adherent cells
cryopreservation of mammalian cell lines

Materials

  • Active, adherent U2OS cells in T-75 flask
    • high confluence is ideal (cells have grown to form a complete monolayer on the flask with few gaps)
  • Active, adherent SKNSH cells in T-25 flask
    • high confluence is ideal (cells have grown to form a complete monolayer on the flask with few gaps)
  • Complete medium for U2OS (McCoy's 5A w/ 10% FBS & 1% pen/strep)
  • Complete medium for SKNSH (Eagle's Modified Essential Medium w/ 10% FBS & 1% pen/strep)
  • 1x phosphate-buffered saline (PBS)
  • trypsin-EDTA
  • T75 flasks
  • 6-well plate for transfections
  • cryo vials for cryopreservation
  • pipette tips
  • 70% ethanol in spray bottle

Notes

U2OS going into 3 vessels:
1. 1:10 passage into T-75 for cryo
2. 1:10 passage into T-75 for backup
3. 6-well plate (final conc. 2E5 cells/well, 4mL per well)

For the 1:10 passages, 9mL medium + 1mL cells

For the 6-well plate:

  • 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



Results/Conclusions

SKNSH culture is labeled PS-β and is in the upper incubator, middle shelf.
U2OS flasks (2) and 6-well plate (1) are in the upper incubator, middle shelf.
U2OS cryo stocks will be prepped later in the week from the PS-1 flask.
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