User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/03: Difference between revisions

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==mm/dd/yy==
==06/03/13==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
* Flow cytometry of PcTF+ U2OS cells


----
----
'''Line item 1'''<br>
'''Flow cytometry at Biodesign'''<br>
> Samples


{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
Samples:
|-valign="top"
# 2.0 μg plasmid/ 1x10<sup>6</sup> cells
| <u>Reagent</u> || <u>Volume</u>  
# 1.0 μg
|-
# 0.5 μg
| reagent 1 || # μL
# 0.2 μg
|-
# 0.1 μg
| reagent 2 || #
# blank (no transfection)
|-
| reagent 3 || #
|-
| reagent 4 || #
|-
| dH<sub>2</sub>O || #
|-
| &nbsp; || # μL
|}


--> Reaction conditions
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'''<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).




Revision as of 15:48, 11 June 2013

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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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