User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/03

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06/03/13

  • Flow cytometry of PcTF+ U2OS cells

Flow cytometry at Biodesign

Samples:

  1. 2.0 μg plasmid/ 1x106 cells
  2. 1.0 μg
  3. 0.5 μg
  4. 0.2 μg
  5. 0.1 μg
  6. blank (no transfection)

Harvesting:

  • Detached cells w/ 0.5 mL trypsin medium
  • Pelleted in ab-free medium
  • Washed once with 1xPBS buffer. Resuspended in 0.5 mL 1xPBS

Flow cytometry:

  • Took cells over the BDI on ice. Strained cells once through cell strainer caps (purchased 10 from Tong Xu); forced cells through cap with pipette (did not centrifuge)
  • Ran samples 6 (blank), 1, 2, and 3 and recorded SS, FS, and mCherry
  • Very modest mCh over background (blank). Tong noted that the mCherry laser was not optimal for this fluorophore.

Flow cytometry - Wang Lab's Accuri C6

  • Diluted samples 3x to get reads per sec. down to 240
  • Signal over background looked pretty good for mCherry
  • Drawback is that voltage/ sensitivity cannot be adjusted, so blank signal is in the 103 range.
  • Do further tests to see if Accuri GFP settings can detect CFP (AmCyan).


Reagent Volume
reagent 1 # μL
reagent 2 #
reagent 3 #
reagent 4 #
dH2O #
  # μL

--> Reaction conditions



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