Cytometry FACS Advanced Technology Laboratory

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Cytometry (FACS)Advanced Technology Laboratory

• FACS Team
• Frank
• Viki
• Sandy
• Padmini (on leave)
• Sophie
• Lankesha
• Angela
• Dora
• David

http//www.wehi.edu.au/cytometry/presentations.htm
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How Flow Cytometry Works

• Structures on or within a cell are marked with
  fluorescent tags.
• Single cells suspended in saline are streamed
  past a focused laser beam.
• Laser light excites fluorescence and scatters in
  all directions.
• Detectors receive flashes of fluorescent and
  scattered light that are processed and
  interpreted by computer.

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Flow Cytometry Multi-colour Detection
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Scattered Light Interpretation

• FSC detectors correlate with cell size
  (cross-sectional area, refractive index).
• SSC detectors indicates nuclear shape and
  cytoplasmic granularity.

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Flow Cytometry and Sorting Schematic
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Questions Answered by Flow Cytometry

• What kind of cell?
• Whats it up to?
• Whats its history?
• And..

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What Kind of Cell?

• Immunofluorescence
• Specific antigen
• Genetic reporters
• FACS Gal
• FISH
• Fluorescent Protein

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What Kind of Cell?

• Immunofluorescence
• Specific antigen
• Genetic reporters
• FACS Gal
• FISH
• Fluorescent Protein

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What Kind of Cell?

• Immunofluorescence
• Specific antigen
• Genetic reporters
• FACS Gal
• FISH
• Fluorescent Protein

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What Kind of Cell?

• Immunofluorescence
• Specific antigen
• Genetic reporters
• FACS Gal
• FISH
• Fluorescent Protein

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Whats the cell up to Quiescent, Activated,
Apoptosing?

• Total DNA Cell Cycle
• RNA Synthesis
• Mitochondria
• Intra-cellular Ca
• Intra-cellular pH
• Phosphatidyl Serine Exposure

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Whats the cell up to Quiescent, Activated,
Apoptosing?

• Total DNA Cell Cycle
• RNA Synthesis
• Mitochondria
• Intra-cellular Ca
• Intra-cellular pH
• Phosphatidyl Serine Exposure

G0/1
G2/M
S
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Whats the cell up to Quiescent, Activated,
Apoptosing?

• Total DNA Cell Cycle
• RNA Synthesis
• Mitochondria
• Intra-cellular Ca
• Intra-cellular pH
• Phosphatidyl Serine Exposure

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Whats its history?

• Time course
• BrdU
• Topical staining
• Tracking

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Whats its history?

• Time course
• BrdU
• Topical staining
• Tracking

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Whats its history?

• Time course
• BrdU
• Topical staining
• Tracking

Cell divisions
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CFSE Fluorescence
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And..

• Cytotoxicity
• Chromosomes
• Parasites
• ...

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Instruments at WEHI

• SORTERS
• FACStar
• FACSVantageSEDiVa
• MoFlo
• FACSAria (2)
• ANALYSERS
• FACScan (2)
• FACSCalibur (2)
• LSR I
• LSR II (2)

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WEHI Instruments Sorters

• Run by FACS lab staff 1015-1700 working days
  only
• Special arrangements over lunch and after 1700
• DIY operation after hours by trained, experienced
  users

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Booking of Time Sorters

• Consult lab staff or go online to discover
  suitable vacancies already on the schedule.
  Requests are vetted by lab staff.
• If there are no current vacancies, fill in a
  paper form or go online to request time 3 weeks
  in advance (http//unix44.alpha.wehi.edu.au/facs_b
  ooking/lab/).
• Advance schedules are set by Monday morning
  lottery.
• Time allocation is qualified-random (ask for
  details).

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WEHI Intruments Analysers

• Do-It-Yourself analysis, available anytime.
• Training provided as well as troubleshooting and
  data interpretation assistance on request.

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Training DIY Flow Cytometry

• Step 1 Multi-media interactive computerised
  training for basic cytometry which is advised
  also for those planning cell sorter use. (enquire
  at FACS Lab).
• Step 2 Group tutorials offered from time to time
  (look for intranet notices)
• Thereafter, individual supervision (by your
  supervisor or by FACS lab staff) with your first
  and subsequent flow cytometric analyses should be
  sought.
• Prospective LSR II users are advised to become
  familiar first with FACScan/FACSCalibur
  operation.
• Data analysis demonstrations and advice can be
  sought at any time.

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Booking of Time Analysers

• Go online up to 3 weeks in advance
  (http//unix44.alpha.wehi.edu.au/facs_booking/user
  /).
• Booking is instantaneous.
• Cancellations more than 24 hrs in advance attract
  no penalty.

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FACS Data Analysis

• Stored FACS data from sorters and analysers may
  be analysed and displayed on PC or Macintosh
  using WEHI's "WEASEL" program.
• Specialist DNA analyses are performed using
  "ModFitLT" one copy of which is available at
  WEHI.
• Specialist cluster analysis is performed using
  "ACluE", available on request for PC or Macintosh
  .

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Bio-safety

• Cell sorters produce aerosols so identification
  of biohazardous samples is compulsory prior to
  booking.
• Biosafety committee approval is required before
  initiating projects involving analysis or
  sorting of samples with a known or potential
  biohazard, including all un-fixed human cells.
• Human samples will be sorted only on selected
  cell sorters in the Cytometry Lab.
• FACS analysers must be decontaminated after use
  to avoid cross contamination (see "rules of
  operation").

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Cytometry (FACS)Advanced Technology Laboratory

• FACS Team
• Frank
• Viki
• Sandy
• Padmini (on leave)
• Sophie
• Lankesha
• Angela
• Dora
• David

http//www.wehi.edu.au/cytometry/presentations.htm
l