Blue:RNA-Seq Experiments:07302013: Difference between revisions

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*Obtained PI stained nuclei from Gwen and Yun
*Obtained PI stained nuclei from Gwen and Yun
*FACs sorted into 10ul in 96-well plate:
*FACs sorted PI positive and FSC high (i.e. largest) population into 10ul FACS buffer in 96-well plate:
:#4000 nuclei - too low nuclei count
:#6000 nuclei - too low nuclei count
:#8000 nuclei - 120,000/ml
:#12000 nuclei - too low nuclei count
:#20000 nuclei - 30,000/ml
:#12000 nuclei in 2ul - 20,000/ml
*Used 10um beads on microscope - confirmed that nuclei are slightly larger than 10um (average of 12um)
*Used sort #3 - 120 nuclei/ul (added 6ul nuclei + 4ul C1 cell suspension solution - loaded 5ul for approximately 360 nuclei total)
*Captured approximately 1/4 of plate - several capture sites showed nuclei clumps  - therefore there are issues with cell clumping and loss of sample after sorting...
*Loaded and continued C1 reaction with captured wells
 
 
FACS Sort:
 
[[File:nuclei.jpg]]

Latest revision as of 01:54, 10 August 2013

Purpose[edit]

  • Fluidigm C1 on human cadaver nuclei (J. Chun Lab)


Experiment[edit]

  • Obtained PI stained nuclei from Gwen and Yun
  • FACs sorted PI positive and FSC high (i.e. largest) population into 10ul FACS buffer in 96-well plate:
  1. 4000 nuclei - too low nuclei count
  2. 6000 nuclei - too low nuclei count
  3. 8000 nuclei - 120,000/ml
  4. 12000 nuclei - too low nuclei count
  5. 20000 nuclei - 30,000/ml
  6. 12000 nuclei in 2ul - 20,000/ml
  • Used 10um beads on microscope - confirmed that nuclei are slightly larger than 10um (average of 12um)
  • Used sort #3 - 120 nuclei/ul (added 6ul nuclei + 4ul C1 cell suspension solution - loaded 5ul for approximately 360 nuclei total)
  • Captured approximately 1/4 of plate - several capture sites showed nuclei clumps - therefore there are issues with cell clumping and loss of sample after sorting...
  • Loaded and continued C1 reaction with captured wells


FACS Sort:

File:Nuclei.jpg