FACS Flow Cytometry Workshop

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FACS Flow Cytometry Workshop
BIOPOLIS Flow Cytometry Facility

• Biopolis
• Singapore
• 05 June 2007
• John Daley
• john_daley_at_dfci.harvard.edu

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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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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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AGENDA

• Introduction Instrument Overview
• 4 Way Sorting
• SP Analysis on murine bone marrow
• Ca flux on murine splenocytes
• Case studies in FACS analysis and sorting
• Discussion

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FACSAria StartupProcedures

• Remove protective Cover
• Lift up Flow cell access door
• Turn on Aria and laser
• Wait for Acquisition dashboard to connect to
  instrument
• Run instrument start up (do long clean once a
  week for sterilization of lines and plenums)
• Turn on stream should look like day before
• Lower flow cell access door
• Turn on sweet spot
• Warm up for 5-10 minutes
• Check sheath fill if necessary
• Change waste tank with back up tank

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General Sort Set Up

• Open Accudrop experiment ( preset experiment or
  new experiment template)
• Run Accudrop
• note FSC-A and FSC-H/SS-A and SS-H/and APC-Cy7
  A and APC-CY7-H note position using title
  letter positions adjust area scaling if necessary
• Adjust flow rate to 2000-2800/sec with setting no
  greater than 3
• Note location of beads with respect to preset
  regions adjust red laser time window to maximal
  signal. Go below and above, note starting time
  value
• Set P1 region on FSC-A histogram to include
  entire scatter region, verify that sort mode is
  set to fine tune and press sort button and press
  cancel for waste drawer to open
• Press optical window button and adjust sort
  timing so that at least 95 of beads fall in left
  sort box on stream camera display
• Note drop 1 value and place in box on droplet
  stream area settings
• Unload tube and run facs clean for 1-2 minutes to
  sterilze for next sort

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FACSAria 4 Way Sorting

• Verify 4 good side streams deflecting to each
  tube. If not fine adjust tip to get satellite
  merger, clean plates, nozzle tip area, strobe
  area and nozzle tip with ddH20- dry with
  q-tip/kimwipe and q-tip static guard to remove
  residual saline, replace o-ring if leaky. (Drop
  pressure by 10 pounds in emergency and reset drop
  delay and timing windows if outer side streams
  can not deflect far enough out)
• Run bead test sort if necessary
• Run mouse spleen spike bead test sort if
  necessary
• Actual Sort Run freshly PRE-FILTERED sample to
  get adequate distribution profile Set up
  template, remove unnecessary parameters, run auto
  comp if necessary, adjust Area scaling , gate out
  doublets, set non-overlapping sort windows, use
  population hierarchy to confirm appropriate
  subpopulation derivation, put windows in sort box
  set mode to preset optimal sort settings(0 32 0)
  turn on test streams, verify stable and well
  separated, turn off test streams, adjust flow
  rate to 1, press sort and listen for waste drawer
  to open. Verify drawer open if not certain. Put
  in appropriate tubes (e.g. eppendorf, or 5ml
  culture tubes)
• Once sorting begins verify side streams on camera
  , temperature set to desired value, sample
  agitation on, adjust droplet charging if
  necessary to get tightest center stream,monitor
  gap value for stability, increase flow rate
  gradually by 1-2 unit increments to desired flow
  rate, note flow rate versus coincidence rate,
  adjust to get maximal yield with minimal
  coincidence adjust window extension width lower
  to get less electronic aborts . Ideal rates for
  purity and sorting and reasonable coincidence
  19-23K/sec
• Note ratio of each 4 side streams , (70u tip with
  above sort precision sorts at a volume of 1
  million per ml), remove most frequent population
  when tube is about 85 full , do while sort is
  going lift flow nozzle access door to stop
  streams temporarily. Replace tube as quickly as
  possible, lower door and resume sorting
• Monitor drop stability, sample volume and side
  streams. Adjust sort gating regions if necessary.
  Listen for spark gap arcing within deflection
  plate area, as well as center stream drift
  outside waste catcher
• Monitor flow rate, if drops down up rate to 11
  and back down to 4-5 see if rate is restored, if
  not unload sample, PRINT SORT REPORT, revortex
  sample, look for clumps, filter if necessary and
  reload sample with flow rate of 1. Reactivate
  sort and increase flow rate gradually back to
  previous flow rate.
• Run sample till no volume left, print sort
  report, rinse lines, and reanalyze sorted
  fractions if desired (recommended)

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4 way Bead sort to check Instrument Accuracy
Protocol

• Beads needed
• 1 Accudrop cat345249
• 2 Calbrite APC cat 340487
• 3 Sphero Rainbow Fluorescent particles 
  3.0-3.4um(mid range FL1) cat 556298
• 4   "           "                "              
  "            "          ( brighter?)     cat
  556291
•  
•  
• First click on new experiment icon and used
  default pmt setting 250, 300, 500,etc.....
• and made single graphs of all pmts  log  except
  FALS and two parameter of Hoechst blue vs. APC
  Cy7
•  
• Second Add a few drops of each bead to separate
  tube add 1xpbs(200ul)    and run/
  record 5,000-10,000events
•  
• Third     Adjust  APC Cy7 down to 385 volts when
  run Accudrop  to get beads on scale. Use
  Biexponential display option
•  
• Four mix beads together one at a time and run
  and gate on where peak showed up on apccy7 vs.
  hoechst blue histogram (that way can see where
  each bead  size scattered  based on color gating)
•  
• Fifth mixed all beads together and create  
  four  distinct sort regions on  APC-CY7 vs.
  Hoechst Blue graph and did a four way sort for
  about 4 minutes sorted about 1.2 million.  Use
  custom sort precision settings which is  0 32 0
  (VERY VERY IMPORTANT!) I did not have them very
  concentrated and sorted at 11 flow rate (bad for
  core stream)
•  reanalyze each fraction with a pbs wash between
  each tube.   

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Simple Four Way bead Sort
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4 way bead sort Spiked with Fixed Murine Spleen
cells
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Murine Hematopoietic Stem Cell/Progenitor Sort
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SP Analysis and Sorting

• Create SP experiment template Hoechst
  blue/Hoechst red/PI/FA/FSC/and SSC with
  appropriate single and two parameter histograms
  including all Area and H parameters. FSC and SSC
  width parameters also selected. If phenotyping is
  desired, select appropriate fluorescent
  parameters. Check that 450 and 670 filters are in
  Trigon and 635lp dichroic filter is in slot. (
  Initially)Make sure signal processing is going to
  correct pmt, test by removing filter and noting
  signal response.
• Run UV bead standard, verify median fluorescence
  intensity and CV value from previous bead
  standard run and preset region window values,
  check UV laser dealy time window verify optimal
  timing by going up and down from preset value by
  2-4 units. Note starting time window value prior
  to changing. Adjust if necessary.
• Recheck Sort delay with Accudrop and do a test
  sort with Accudrop and uv bead mix to verify UV
  channel is being delayed correctly ( usually not
  necessary). Make sure all systems are ice cold.(
  sample station and collection holder)
• Run SP controls and sample . Adjust Scatter and
  Fluorescent PMT voltages so that scatters are on
  scale and controls are set to appropriate scale
  location. Use log Ho blue and red to see overall
  DNA cell cycle distributions, check UV timing
  window and see if increased signal can be
  observed by increasing window timing slightly, if
  not return to original setting. Gate on PI
  negative population and do a drill down to a HO
  blue (y Axis) vs. HO red (Y axis) profile .
  Switch HO BLUE to Linear and adjust voltage so
  that G0/G1 peak is at channel 100,000 note cell
  cycle profile Increase HO BLUE PMT voltage so
  that G0/G1 linear HO-Blue peak is centered up
  around channel 200,000 and G2/M peak is off
  scale. Adjust HO-Red sample so that its G0/G1
  peak position is also around 200,000 or so. Let
  Stream stabilize at lower flow rate settings(1-2)
  for a minute or two to counter effects of
  initial sample boost., then record at least
  100,000 PI negative gated events. Repeat with
  verapamil or reserpine control samples. Look for
  SP tail or hook below and slightly to left of
  G0/G1 cluster. Better to adjust concentration
  than to raise flow rate value. Aliquot sample in
  multiple small tubes
• If population is identified set up sort gates for
  sp and non- SP areas and sort. Small eppendorf
  vial collection tubes pre-coated and filled
  30-65 with appropriate media is recommended.
  Remember sorted concentration is approximately
  one million/ml so be sure to not overfill
  collection vessel.
• Reanalysis of sorted population can be conducted
  if desired. Note however that sorted fractions
  may pump out Hoechst dye while being sorted and
  will also lose dye while being collected in a non
  Hoechst containing media collection vial.
• Check with investigator about how cells were post
  sort and what results were obtained to aid in
  improving future sorts

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UV Bead StandardFluoresbrite BB Carboxylate
Microspheres 4.5µmPolysciences
LOG
LINEAR
Hoechst Blue PMT 225 Volts
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FACSARIA SORPUV LASER
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Calcium Flux on Murine Splenocytes

• Check Filters on UV Trigon Indo Violet405nmBP,
  Indo Blue530nmBP with 505LP dichroic
• Run UV bead standard adjust timing if necessary
• Set up Calcium flux experiment template. Use FSC
  vs. SSC/SSC vs., Time versus ratio (25,50 and
  75),Indo Blue vs. Indo Violet (LOG vs. LOG),
  relevant Immunofluorescent Histograms and time
  vs. ratio of subpopulations if necessary
• Check to see that sample station temperature is
  set to Off, 20C, or 37C depending on desired
  temerature. Check to see sample agitation is set
  to 300 rpm
• Run controls( unstained, indo alone, single
  color, etc) set up compensation, adjust ratio so
  that resting population 25 ratio is at 40,000
  to50,000 value. Use worksheet histogram zoom in
  to zoom in on time versus ratio histogram
• Actual Flux RUN set stopping time to 30
  seconds, set events to recort to 1million or 2.5
  million, load indo1 loaded sample, after boost
  waitfor 5 seconds or so and press record, after
  record is done, unload sample, LIFT NOZZLE ACCESS
  DOOR, RESET TIME TO 270 SECONDS,ADD
  STIMULI,VORTEX BRIEFLY,LOAD SAMPLE ,, PRESS
  RECORD, PRESS APPEND, COUNT 5 SECONDS, LOWER
  NOZZLE ACCESS DOOR, and continue run.
• Sit back and relax
• Transfer files to post analysis program to
  calculate percent responding and relative
  increase in calcium concentration. Use FloJo ,or
  create regions using Diva software and statistics
  export in Excel spreadsheet

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Ca flux with Indo 1-AM
Useful to study heterogeneity within Defined
subpopulations

• -Ratio metric Dye Normalizes for cell size
  variability
• As well as laser power fluctuations over time
• -Very sensitive to slight shifts within
  populations
• Ultraviolet excited can be used with many visibly
  excited
• fluorochromes

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Calcium Flux

• Method to get Gap in Time vs. ratio Histogram (
  Lift Cover/reset time stop/append file/close
  cover after 5 seconds)

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FACSAria UV ApplicationsCalcium Flux Kinetic
Assays
Ionomycin
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Calcium Flux on T cell subpopulations
FACSAria SORP 20Mw 355nm UV Laser
10ug GAM IgG1
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Case Studies in FACSAnalysis and Sortingto be
provided by participants

• Actual samples
• Virtual samples
• Viable Cell Cycle with HO33342 on dual stained
  GFP/DsRed cells
• Treg Various compensation methods
• Biexponential data displays

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GFPDsRedHoechst 33342Viable Cell Cycle
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Fluorescent proteins
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Multiple Gating strategies help identify low
frequency functional subpopulations
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Multicolor(5) Minor populations Four way
sorting Treg Story
CD45ra FITC CD127 PE CD25 PE-CY5 CD4 PE-CY7 CD3
PAC BLUE
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Treg Post Sort Reanalysis
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POST SORT REANALYSIS HUMAN PBL CD3CD4 CD45RA
CD45RA-
Treg/Teffector/ --
98.4
99.4
97.1
99.9
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Discussion

• Instrument Specific
• Essentials
• Clog Control
• Flow Cytometry in General
• Anything else

THANK YOU!
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FACSAria Essentials
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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