User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/04

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07/04/13

  • Gl4-EED/luc time/dose - luc assay & cell counts
  • Happy July 4th! :)

Luciferase activity assay - time point: 4 days


Cell prep

  • Harvested cells (induced on 6/30/13)
    • Seeded new 2 new plates with non-induced cells from 6/30/13
    • Pelleted all of the dox+ cells, 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
  • Used opaque white Costar plate (from Rege lab)
  • Samples loaded in triplicate (by columns)
  • Included luc buffer + FACS-buffer "blank" sample (well D1)
  • 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 ### 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