refresher: conventional flow, spectral flow, cytof
TRANSCRIPT
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Lisa BorghesiAssociate Professor of Immunology
Director, Unified Flow Core
Refresher:Conventional Flow, Spectral Flow, CyTOF
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Conventional flow cytometry“one detector, one color”
PMT 1
PMT 2
PMT 5
PMT 4
DichroicFilters
BandpassFilters
Laser
Flow cell
PMT 3
forwardscatter
Sample
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From Practical Flow Cytometry Third Edition, Howard M. Shapiro p.164.
Spectral Overlap
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Our high speed analyzers
3 Cytek Auroras (2 in Unified Core, 1 at HCC)= spectral
3 BD LSRFortessa, 2 BD LSRII, 1 NxT Attune= conventional
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Spectral flow cytometry: Cytek Aurora
collect ALL of the light àincreased sensitivity
discriminate fluors with overlapping spectra à not
possible with conventional flow cytometry
64 channels
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FACSAntibodies labelled
with fluorochromes
CyTOFAntibodies labelled with
elemental isotopes
Conventional cytometry, 10-15 colors; beyond that
gets tough
Spectral technologies = game changer, 64
channels
40-50 parameters in a single tube
Ex. gadolinium (Gd), samarium (Sm), terbium
(Tb), thorium (Th), ytterbium (Yb)
Spectral cytometry rivals CyTOF capabilities
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Pitt Aurora Flow Panels Repository
Thanks to our users for sharing their panel designs!
• Panels for each mouse and man• Designed/validated by our Pitt colleagues• Excel spreadsheet located in same PittBox that contains
this ppt
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Next Tutorial: Harness the Full Information Content of Your Data with
Algorithmic Analysis
• High-dimensional analysis – Human brain is not so hot at >3 dimensions.
• Highlight patterns in the data – Manual analysis with iterative bivariate plots is highly inefficient.
• Facilitates population discovery –Populations can be overlooked b/c biases and a priori knowledge dictate analysis.
• Fun!
tSNE_X
tSN
E_Y
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Acknowledgements
Dewayne FalknerDirector of Operations
Aarika MacIntyreSenior Technologist
Tim SturgeonSenior Flow Cytometry Specialist
Unified Core, BoardMark ShlomchikFadi LakkisMark GladwinLarry MorelandJohn McDyer
Nan ShengCore Technician
Heidi GunzelmanSenior Flow Cytometry Specialist