Tissue Culture Training
- Passaging - U2OS, split PS3 --> PS4
- 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: 4
- 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
- Passaging - KAH126, split PS3 --> PS4
- 6-well plate - KAH126
- Passaging - KAH126
- 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: 4
- Dox induction - KAH126
- Induce cells in 6-well plate with dox to express PcTF (RFP)
- Stock concentration of dox = 1.0 mg/mL
- Desired final concentration of dox = 1.0 μg/mL (1000x diluted)
- Therefore add 1.0 μL of stock dox for every 1.0 mL medium in well
- 4.0 mL medium in well x 1.0 μL stock dox = 4.0 μL stock dox to be added to each well
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