01/28/15
Cytology of KAH126
Cell plating (1/23/15)
- Poly lysine coating of cells (done last week) - follow 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)
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 protocol
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