User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/03: Difference between revisions
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* | * Flow cytometry of PcTF+ U2OS cells | ||
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''' | '''Flow cytometry at Biodesign'''<br> | ||
> | |||
Samples: | |||
# 2.0 μg plasmid/ 1x10<sup>6</sup> cells | |||
# 1.0 μg | |||
# 0.5 μg | |||
# 0.2 μg | |||
# 0.1 μg | |||
# 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. | |||
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'''Flow cytometry - Wang Lab's Accuri C6'''<br> | |||
* 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 10<sup>3</sup> range. | |||
* Do further tests to see if Accuri GFP settings can detect CFP (AmCyan). | |||
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Revision as of 15:47, 11 June 2013
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06/03/13
Flow cytometry at Biodesign Samples:
Harvesting:
Flow cytometry:
Flow cytometry - Wang Lab's Accuri C6
--> Reaction conditions
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