Haynes Lab:Notebook/Jan/2015/08/31: Difference between revisions

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(Autocreate 2015/08/31 Entry for Haynes_Lab:Notebook/Jan)
 
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==Entry title==
==Cell Culture==
* Insert content here...
* Passaging - U2OS, split PS5 --> PS6
*6-well plate - U2OS
*Passaging - U2OS
*Protocol: [http://openwetware.org/wiki/Haynes:SplittingCells Splitting Cells]
*Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S
*Use 90-100% confluent T-75
*Make 1 new T-75 at a 1:10 dilution
*Passage number: 5


* 6-well plate - U2OS
* Use same resuspension from old flask in Passaging - U2OS
*Starting with: 10E6/10 mL = 1.0E6/1 mL stock cell resuspension concentration
*Desired cells/ well in new 6-well plate = 2.5E5 (250k) cells in 4.0 mL medium
*Amount of starting culture(x) to use: x = (1.0E6 cells / 1 mL) / 2.5E5 cells = 0.25 mL stock cell resuspension per well
*Amount of fresh medium to use: 4.0 mL final volume - 0.25 mL stock cells = 3.75 mL fresh medium per well
*Make a batch of diluted cells for new 6-well plate:
*Into a sterile 50 mL conical add...
*(6 wells + 1 pipetting error) x 0.25 mL stock cells = 1.75 mL stock cells
*(6 wells + 1 pipetting error) x 3.75 mL fresh medium = 26.25 mL fresh medium


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Revision as of 13:28, 31 August 2015

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Cell Culture

  • Passaging - U2OS, split PS5 --> PS6
  • 6-well plate - U2OS
  • Passaging - U2OS
  • Protocol: Splitting Cells
  • Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S
  • Use 90-100% confluent T-75
  • Make 1 new T-75 at a 1:10 dilution
  • Passage number: 5
  • 6-well plate - U2OS
  • Use same resuspension from old flask in Passaging - U2OS
  • Starting with: 10E6/10 mL = 1.0E6/1 mL stock cell resuspension concentration
  • Desired cells/ well in new 6-well plate = 2.5E5 (250k) cells in 4.0 mL medium
  • Amount of starting culture(x) to use: x = (1.0E6 cells / 1 mL) / 2.5E5 cells = 0.25 mL stock cell resuspension per well
  • Amount of fresh medium to use: 4.0 mL final volume - 0.25 mL stock cells = 3.75 mL fresh medium per well
  • Make a batch of diluted cells for new 6-well plate:
  • Into a sterile 50 mL conical add...
  • (6 wells + 1 pipetting error) x 0.25 mL stock cells = 1.75 mL stock cells
  • (6 wells + 1 pipetting error) x 3.75 mL fresh medium = 26.25 mL fresh medium


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