Blue:RNA-Seq Experiments:07302013: Difference between revisions
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>B1lake (Created page with "== Purpose == *Fluidigm C1 on human cadaver nuclei (J. Chun Lab) == Experiment == *Obtained PI stained nuclei from Gwen and Yun *FACs sorted into 10ul in 96-well plate:") |
>B1lake No edit summary |
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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:
- 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: