User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/19

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06/19/13

  • Luc assay, time point 2
  • PcTF transfection for U2OS, outgrowth #2

Luciferase activity assay - time point 2


Cell prep

  • Harvested cells
    • Seeded new dox+ plate with 1/2 of cells
    • Pelleted other half, resuspended in 2 mL FACS buffer


Assay reagents

  • Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as 6/18/13)


Luc assay

  • Filtered 700 μL cells through strainer caps
  • Other steps same as 6/18/13


Cell counts

  • Wang lab's Accuri flow cytometer.
  • Set machine to read 20 uL of cells
  • Be sure to "clean" with water-run in between samples


Sample ID Gated count/ 20 μL   Cells/ 100 μL
sample 1 10,880 x5 = 54,400
sample 2 10,430 x5 = 52,150
sample 3 11,897 x5 = 59,485
sample 4 10,316 x5 = 51,580
sample 5 9,784 x5 = 48,920
sample 6 11,549 x5 = 57,745



PcTF transfection for U2OS, outgrowth #2

  • Expanded transfected and mock-transfected cells from old 10 cm plates
  • 1:10 passage of PcTF+ or mock cells into three new 10 cm plates labeled "A, B, C" (standard complete medium)



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