User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/18

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06/18/13

  • Luciferase activity assay



Luciferase activity assay - time point 1


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
  • Thawed reagents to room temp
  • 6 experimental samples x 3 repliates = 18 assays (one time point)
  • Need 100 μL complete steady-luc buffer per assay = 1800 μL complete steady-luc buffer
  • Dissolved dry D-luciferin to make 50x stock by adding 80 μL dH2O to the vial


Luc assay

  • Filtered 500 μL cells through strainer caps (should use 600 μL next time)
  • Transferred 100 μL strained cells to 3 wells in 96-well plate (black walls; clear, flat bottom)
    • Saved remainder of strained cells for flow cytometry/ counting
  • Made 2000 μL complete steady-luc buffer: Added 40 μL 50x D-luciferin to 2 mL buffer
  • QUICKLY Added 100 μL of complete steady-luc buffer to each sample (pipetted up/down once to mix)
  • Incubated at room temp/ 10 min.
  • Synergy H1 reader - Created new protocol: KAH_luciferase
    • Optimized specs: Luminescence, Endpoint, Full plate, Integration time 0:01.00 (MM:SS.ss), Filter set 1, Emission: full light, Optics: top, Gain 200, Read Speed: normal, Delay: 100 msec, Extended Dynamic Range, Read Height: 1 mm


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 8077 x5 = 40,385
sample 2 9552 x5 = 47,760
sample 3 9460 x5 = 47,300
sample 4 3608 x5 = 18,040
sample 5 4723 x5 = 23,615
sample 6 5851 x5 = 29,255



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