Flow Cytometry and the FACSAria

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Flow Cytometry and the FACSAria
Singapore 2007

• Teaching a new dog old tricks

John Daley john_daley_at_dfci.harvard.edu
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Basics of Flow
(modern day alchemy)

• Air
• Fire
• Water
• INTELLIGENCE!

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1977 vs. 2007

• Air house 12lbs
• Fire 500mW 488nm Blue laser H20 cooled LARGE
• Water 1 liter 1xPBS
• Jet in Air Nozzle (circle of fire)

• Air self made 70lbs
• Fire3-7 low power lasers from UV to deep red/
  Air cooled small/
• Water 10 Liter dual 0.22u filtered PBS
• Fixed Cuvette Nozzle

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What hasnt changed

• Laser stream intersection
• Cell interrogation via hydrodynamic focusing
• Droplet formation for sorting
• Priority for cellular integrity

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What has changed

• Standard Multi Laser Excitation
• Polychromatic Analysis
• High Throughput 4 way cell sorting
• Instrument automation routines
• Expansion of applications
• Instrument sophistication

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FACSAria
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FACSAria
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Workshop Objectives

• Share hands on experiences for users and
  operators
• Highlight major elements of FACSAria
• Optimize instrument utilization
• Verify Experimental Set-ups
• Minimize instrument down time
• Have fun and learn something

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Selected Applications

• Stem cell subpopulations
• HSC/PRO CMP MEP GMP
• Compensation issues
• UV Applications
• Hoechst 33342
• Ca Flux
• DAPI
• Q-dots
• Four way sorting
• Tube selection
• Coincidence use
• Single cell Deposition
• Terasaki plates
• 384 well plates
• PCR plates
• Off line Analysis

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The Chi of Flow Cytometry

• Organization
• Standardization
• Observation
• Implementation
• Instrument ?

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Turning a user into an operator ( and vice versa)

• Fixed Cuvette
• Ceramic Flow Cell
• O ring stabilization
• Clog Control
• Compensation selection
• Compensation correction
• Dynamic Sorting

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Fixed Cuvette/nozzle tip

• Keep windows clean
• Know thy nozzle tip
• Correct droplet strobe pattern
• Backup tip with o-ring
• Proper nozzle reseat techniques
• Camera clean
• Static neutralization
• 70 vs. 100 micron tip

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Compensation Selection

• Why Compensate
• Beads vs. cells
• Auto compensation
• gt100 compensation
• Compensation minimization
• Exceptions to the rules

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Clog ControlLevel I

• Lift Cover
• Open door check for spray
• Instrument clean-gt Contrad 2 times
• Turn on stream
• Door open check for stream
• Check for strobe droplet recovery
• Check for red laser intercept spot
• If none of above go to Level II

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Clog ControlLevel II

• Clean table/ organize tools
• Remove tip/ remove o-ring
• Remove cam/ dry excess fluid
• Visual check tip with 10x obj if poss.
• Ultrasonicate 10 sec minimal ddH20
• Kim wipe/Vacuum air dry tip/ visual check orifice
• Reseat o-ring with q-tip and ddH20
• Place back in nozzle/cam up/ check o-ring
• Turn stream on/ verify Level I recovery steps
• Hand adjust nozzle cam to restore droplet pattern

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Clog ControlLevel III

• Repeat Level I and II for 2-5 minutes
• Replace o-ring
• Repeat Level I and II
• Remove plates
• Remove nozzle/ run pure stream
• Turn off stream/ clean all saline/H20
  rinse/static guard spray/ turn on stream if ok /
  turn off stream/reseat plates/ turn on stream
  apply level I / look for arcing / turn on sweet
  spot/ check test streams/ look for stable strobe
  stream
• Yell at user for not filtering sample
• Re filter sample if visually looks particulate
• Replace nozzle

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Disaster Zone

• Cap on 15 ml tube during loading
• No plates installed during sorting
• Waste catcher not open during sorting
• Bleach carry over
• PMT assignment mismatch
• FACService hacking
• The missing 0-ring
• Laser beam mode loss
• Dried saline Cuvette windows
• Promiscuous beam alignment
• Tubing crush
• Loose deflection plates
• 50 deflection max
• Bleach destruction
• The Flood!

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Future Directions Instrumentation

• Recycling of middle stream (waste)
• Uniform cell alignment
• 532nm and 561nm laser addition
• Variable collection receptacles
• Dual chamber sample station
• Injection module
• Real time compensation adjustment
• Clinical grade cell sorting

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Future Directions Applications

• Two Photon flow cytometry excitation
• Enhanced signal detection via RASC
• FRET using FITCCY5
• Cell Cycle compartmentalization with Fluorescent
  protein subpopulations
• Ca Flux of fluorescent subpopulations
• Spectral balancing of fluorescence
• Q-dots bead coding

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Essential Accessories

• Contrad
• O-ring magnifier
• Ultrasonicator
• Vortexer
• Cell strainers
• UV/Violet Beads
• Cotton tip applicators
• Canned air
• Cell Phone number of BD service specialist

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SORP FACSAria UV Apps.

• Viable cell cycle
• Ca Flux
• DAPI
• Q-Dot

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FACSAria UV Applications
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FACSAria UV Applications
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FACSAria UV Applications
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Multicolor T reg
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