- Flow cytometry of PcTF+ U2OS cells
Flow cytometry at Biodesign
- 2.0 μg plasmid/ 1x106 cells
- 1.0 μg
- 0.5 μg
- 0.2 μg
- 0.1 μg
- blank (no transfection)
- Detached cells w/ 0.5 mL trypsin medium
- Pelleted in ab-free medium
- Washed once with 1xPBS buffer. Resuspended in 0.5 mL 1xPBS
- 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.
- ~1% RFP+ over background (with gating)
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).