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Fluorescence Activated Cell Sorting

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by

Anne W

on 14 November 2013

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Transcript of Fluorescence Activated Cell Sorting

Fluorescence Activated Cell Sorting
Sarah E. Millar, Ph.D.
Department of Dermatology
Perelman School of Medicine, University of Pennsylvania
Anne Wang
Overview
Application to Transgenic Mouse Model
FACS Facts
History
Mechanism
Interesting uses
K10-CFP reporter mouse progress
Cornified

Granular


Spinous


Basal
Triple Fluorescent “Rainbow” Mouse
IVL - TomatoI




Krt10 - CFP


Krt14 - GFP
Fluorescence Activated Cell Sorting
(FACS)

Fluidics System
Optics System
Sheath Flow
Sample
Laser
Side scatter,
Side fluorescence
Forward scatter
(488nm)
(500-560nm)
(555-595nm)
(650-690nm)
(<515nm)
Limitation dependent on availability of laser
FACS Aria
Blue 488nm
Red 633nm
Yellow Green 561nm
Violet 405nm
Preparation for FACS
Fluorescent tagging
High throughput assays
Detection of cellular components
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Digital Analysis
Electric System
Application to Transgenic Mouse Model
Interesting Uses of FACS
Analyze mRNA content of sorted cell population
Separate stem cells in blood transfusion
K10-CFP reporter progress
CT 20x MT 20x MT 40x
Acknowledgments
Millar Lab
Sarah E. Millar, Ph.D.
Fang Liu, Ph.D.
Yeon-Sook Choi, Ph.D.
Mingang Xu, Ph.D.
Early vs Modern FACS machine
ICP 11 (1969)
Phywe Gottingen
First commercial flow cytometer
Analog - processing in circuits
500 eps
FACS Aria (2003)
Becton-Dickinson, trademarked FACS
Digital - processing in computer
20,000 eps
Full transcript