User:Karmella Haynes/Notebook/PcTF Genomics/2015/01/28

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01/28/15

  • Cytology of KAH126

Cytology of KAH126

Cell plating (1/23/15)

  • Poly-L-lysine coating of cells (done last week) - follow Mammalian IFC protocol
  • Seed cells at 100k per well (aliquot in 1mL volumes into 24-well plate with poly-lysine coated cover slips on bottom)
  • Same day - Induce cells with 1.0, 0.1, and 0.01 μg/mL dox; final volume = 2 mL
    • Tube 1: Added 8 μL 1 mg/mL dox to 4 mL growth medium (2x target concentration)
    • Tube 2: 0.5 mL from tube 1 into 4.5 mL medium (")
    • Tube 3: 0.5 mL from tube 2 into 4.5 mL medium (")
    • Added 1 mL tube 1 into 1 mL cells in wells 1 & 2 (dilution by 1/2 --> reached target concentration of dox)
    • Added 1 mL tube 2 into 1 mL cells in wells 3 & 4 (")
    • Added 1 mL tube 3 into 1 mL cells in wells 5 & 6 (")
    • Wells 7 & 8 got 1 mL medium without dox


Expression - next day (1/24/15)

  • No signs of PC speckles


Expression - 5 days post induction (1/28/15)

  • Neglected the cells, checked today out of curiosity; growth medium still healthy color
  • A few cells show what look like Pc speckles in the nucleus; most others have diffuse RFP
  • Fix and permeabilize these for IF staining - follow Mammalian IFC protocol



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