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

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(Autocreate 2013/06/03 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
Current revision (17:48, 11 June 2013) (view source)
(06/03/13)
 
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==mm/dd/yy==
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==06/03/13==
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* Line item 1
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* Flow cytometry of PcTF+ U2OS cells
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'''Line item 1'''<br>
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'''Flow cytometry at Biodesign'''<br>
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> Samples
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{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
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Samples:
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|-valign="top"
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# 2.0 μg plasmid/ 1x10<sup>6</sup> cells
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| <u>Reagent</u> || <u>Volume</u>  
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# 1.0 μg
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|-
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# 0.5 μg
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| reagent 1 || # μL
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# 0.2 μg
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|-
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# 0.1 μg
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| reagent 2 || #
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# blank (no transfection)
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|-
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| reagent 3 || #
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|-
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| reagent 4 || #
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|-
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| dH<sub>2</sub>O || #
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|-
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| &nbsp; || # μL
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|}
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--> Reaction conditions
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Harvesting:
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* Detached cells w/ 0.5 mL trypsin medium
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* Pelleted in ab-free medium
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* Washed once with 1xPBS buffer. Resuspended in 0.5 mL 1xPBS
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Flow cytometry:
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* 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)
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* Ran samples 6 (blank), 1, 2, and 3 and recorded SS, FS, and mCherry
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* Very modest mCh over background (blank). Tong noted that the mCherry laser was not optimal for this fluorophore.
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** ~1% RFP+ over background (with gating)
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----
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'''Flow cytometry - Wang Lab's Accuri C6'''<br>
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* Diluted samples 3x to get reads per sec. down to 240
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* Signal over background looked pretty good for mCherry
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* Drawback is that voltage/ sensitivity cannot be adjusted, so blank signal is in the 10<sup>3</sup> range.
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* Do further tests to see if Accuri GFP settings can detect CFP (AmCyan).

Current revision

Pc-TF Genomics Main project page
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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.
    • ~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).



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