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Cytometry (FACS) Advanced Technology Laboratory
 

Cytometry (FACS) Advanced Technology Laboratory

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  • Flow cytometry is the measurement (meter) of characteristics of single cells (cyto) suspended in a flowing saline stream. A focussed beam of laser light hits the moving cell and light is scattered in all directions. Detectors receive the pulses of scattered light and they are converted into a form suitable for computer analysis and interpretation.
  • Here we see a schematic view of the inside of a cell sorter. You can see the three different lasers at the back,the blue, red and UV lasers. these excite multiple fluorochromes, which are detected by the use of different filters and mirrors. These signals are collected by the detectors and are converted into computer analysis data.
  • The total amount of forward scattered light detected is closely correlated with cell size, whereas the amount of side scattered light can indicate nuclear shape or cellular granularity.Further properties of the cell, can be accurately quantified if the cellular marker of interest can be labeled with a fluorescent dye; for example, an antibody-fluorescent dye conjugate may be used to attach to specific surface or intracellular receptors. Other dyes have been developed which are sensitive to the local chemistry (e.g. Ca++ concentration, pH, etc.).
  • Flow cytometric analysis: This is a very schematic view; of the inside of a cell sorter. The real power of flow cytometry arises from our ability to mark cellular structures and properties with multiple fluorescent tags that can be individually detected. Here we see cells passing through a dstream of saline, within the cytometer, The stream is broken into droplets at a predetermined position and cell sorting is done by an accurately timed charging of each droplet containing the selected cell. As we see here the charged cell is deflected into our collection tubes at the bottom of the cytometer.
  • These analytical functions will be illustrated here using flow cytometry examples, all of which are adaptable to confocal microscopy.
  • Data for mouse spleen stained to detect germinal centre B-lymphocytes. Cocktail is a mix of antibodies binding non-B-cells all bearing the same fluorochrome. Data is presented as “dot plots” where each cell is represented as a dot. The dot colour is assigned by a separate computer program that scans the multi-dimensional data space for clusters of related cells. Clusters cannot be discovered visually because a maximum of 3 parameters can be simultaneously displayed (RHS) and even then the display may not be easily interpretable.
  • Data for mouse spleen stained to detect germinal centre B-lymphocytes. Cocktail is a mix of antibodies binding non-B-cells all bearing the same fluorochrome. Data is presented as “dot plots” where each cell is represented as a dot. The dot colour is assigned by a separate computer program that scans the multi-dimensional data space for clusters of related cells. Clusters cannot be discovered visually because a maximum of 3 parameters can be simultaneously displayed (RHS) and even then the display may not be easily interpretable.
  • Marking B-lymphocytes specific for a particular (fluorescinated) antigen.
  • Most commonly used genetic reporters now encode fluorescent proteins of a number of colours. Excitation and emission characteristics may be widely spaced. Blue and green fluorescent proteins may be excited at 350nm (UV) and 488nm (blue-green) respectively. Combinations can mark the presence of multiple genetic inserts.
  • A cell, beginning from G0 enters the cycle (G1) and then begins DNA synthesis. DNA synthesis (S-phase) then proceeds until all chromosomes have been duplicated (G2 phase) and then division occurs, producing 2 G0/1 cells. The plot represents thousands of cells but the status of any one of those cells is reflected in its position within the plot.
  • Data: Marina Carpinelli, WEHI Ref: Preffer et al. 2002 Stem Cells 20:417-427
  • A cell, beginning from G0 enters the cycle (G1) and then begins DNA synthesis. DNA synthesis (S-phase) then proceeds until all chromosomes have been duplicated (G2 phase) and then division occurs, producing 2 G0/1 cells. The plot represents thousands of cells but the status of any one of those cells is reflected in its position within the plot.
  • A cell, beginning from G0 enters the cycle (G1) and then begins DNA synthesis. DNA synthesis (S-phase) then proceeds until all chromosomes have been duplicated (G2 phase) and then division occurs, producing 2 G0/1 cells. The plot represents thousands of cells but the status of any one of those cells is reflected in its position within the plot.
  • INDO-1 emits 2 colours of fluorescence with relative intensities dependent on local Ca++ concentration. The ratio of these emissions thus indicates Ca++ concentration but also removes any variation due to uneven dye loading. Phenotype can be analysed simultaneously and elapsed time recorded as each cell is detected. A time course for activation of the whole sample is then obtained although each cell is only analysed at one instant.
  • A cell, beginning from G0 enters the cycle (G1) and then begins DNA synthesis. DNA synthesis (S-phase) then proceeds until all chromosomes have been duplicated (G2 phase) and then division occurs, producing 2 G0/1 cells. The plot represents thousands of cells but the status of any one of those cells is reflected in its position within the plot.
  • To these techniques may be added detection of the expression of the cyclins using monoclonal antibodies that now exist and the TUNEL technique that uses terminal deoxynucleotidyl transferase (TdT) for labelling of DNA strand breaks with fluorescent nucleotides.
  • Whole thymus dipped in Hoechst dye marks the outer cortical cells. If thymus is subsequently dispersed and cells are stained for phenotype, the geographical distribution of cell types is obtainable.
  • CFSE binds tenaciously to cells over hours or even days and over several divisions. At each division, the dye is shared between the daughter cells so that fluorescence intensity is a marker of cell divisions undergone.
  • Here at WEHI we have 5 sorters, 4 high speed and 1 low speed, as well as 7 analysers.
  • We have 3 sorters in the FACS lab. These are run from 10:15 - 5pm Monday throu Friday.
  • Booking of the cell sorters is done by consultation with FACS Lab staff. Bookings are made up to three weeks in advance via request forms or online. If an immediate sorting time is required and there are no vacancies you can be placed on a waiting list and advised of any last minute cancellations. It is all users responsibility to check the schedule at the start of each week for their allocated time.
  • Time can be used for cell death assays. Pulse processing is used for doublet detection
  • Booking of the cell sorters is done strictcly by consultation with Facs Lab staff. Bookings are made three weeks in advance via request forms. If an immediate sorting time is required, please come and see facs Lab staff and we will find a vacancy for you. If there are no vacancies you can be placed on the waiting list, and advised of any last minute cancellations. It is all users responsibility to check the booking board at the start of each week for their allocated time.
  • We have several computer progrms available to analyse your facs data. These are Cell quest, weasel,motfitLT, and Aclue. These are all available on computers here at WEHI.
  • Cell sorting produces aerosols and therefore all biohazardous samples must be identified and labelled prior to booking/running. On the Facs analysers cross contamination between uses must be avoided and therefore decontamination procedures must be followed. These can be found in the rules of use pasted in the Facs Scan room No human samples will be run on the 6th floor sorters and should be directed to the third floor aria (see Kent Jenson).

Cytometry (FACS) Advanced Technology Laboratory Cytometry (FACS) Advanced Technology Laboratory Presentation Transcript

  • Cytometry (FACS) Advanced Technology Laboratory
    • FACS Team:
            • Frank
            • Sandy
            • Angela
            • David
            • Hesham
            • Rumbi
            • Dora
            • Padmini
            • Lankesha (on leave)
    http://www.wehi.edu.au/cytometry/presentations.html
  • 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.
  • Flow Cytometry: Multi-colour Detection Cy5.5
  • Scattered Light Interpretation
    • FSC detectors correlate with cell size (cross-sectional area, refractive index).
    • SSC detectors indicates nuclear shape and cytoplasmic granularity.
  • Flow Cytometry and Sorting: Schematic
  • Questions Answered by Flow Cytometry
    • What kind of cell?
    • What’s it up to?
    • What’s its history?
    • And..
  • What Kind of Cell?
    • Immunofluorescence
    • Specific antigen
    • Genetic reporters
      • FACS Gal
      • FISH
      • Fluorescent Protein
  • What Kind of Cell?
    • Immunofluorescence
    • Specific antigen
    • Genetic reporters
      • FACS Gal
      • FISH
      • Fluorescent Protein
  • What Kind of Cell?
    • Immunofluorescence
    • Specific antigen
    • Genetic reporters
      • FACS Gal
      • FISH
      • Fluorescent Protein
    17-colour immunofluorescence: Perfetto SP et. al. 2004 Nat Rev Immunol. 4(8):648-655 Laser Fluorochrome 488nm FITC 532nm PE PE.TR PE.Cy5 PE.Cy5.5 PE.Cy7 633nm APC Alexa 680 APC.Cy7 407nm Cascade Blue AmCyan Qdot 545 Qdot 565 Qdot 585 Qdot 605 Qdot 655 Qdot 705
  • What Kind of Cell?
    • Immunofluorescence
    • Specific antigen
    • Genetic reporters
      • FACS Gal
      • FISH
      • Fluorescent Protein
  • What Kind of Cell?
    • Immunofluorescence
    • Specific antigen
    • Genetic reporters
      • FACS Gal
      • FISH
      • Fluorescent Protein
  • What’s the cell up to: Quiescent, Activated, Apoptosing?
    • Total DNA: Cell Cycle
    • RNA Synthesis
    • Mitochondria
    • Intra-cellular Ca ++
    • Intra-cellular pH
    • Phosphatidyl Serine Exposure
  • What’s 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 S G2/M
  • Identification via a Functional Marker: Hoechst 33342 Hoechst Red Hoechst Blue Sca-1.FITC C-kit.Cy5 C-kit.Cy5 Sca-1.FITC SP Cells
    • Total DNA: Cell Cycle
    • RNA Synthesis
    • Mitochondria
    • Intra-cellular Ca ++
    • Intra-cellular pH
    • Phosphatidyl Serine Exposure
    What’s the cell up to: Quiescent, Activated, Apoptosing? RNA DNA Hoechst vs Pyronin Y Ref: Shapiro HM Cytometry. 1981 2 :143-150
    • Total DNA: Cell Cycle
    • RNA Synthesis
    • Mitochondria
    • Intra-cellular Ca ++
    • Intra-cellular pH
    • Phosphatidyl Serine Exposure
    What’s the cell up to: Quiescent, Activated, Apoptosing? forward scatter Rhodamine123 SCA-1 lineage cocktail
  • What’s the cell up to: Quiescent, Activated, Apoptosing?
    • Total DNA: Cell Cycle
    • RNA Synthesis
    • Mitochondria
    • Intra-cellular Ca ++
    • Intra-cellular pH
    • Phosphatidyl Serine Exposure
    • Total DNA: Cell Cycle
    • RNA Synthesis
    • Mitochondria
    • Intra-cellular Ca ++
    • Intra-cellular pH
    • Phosphatidyl Serine Exposure
    What’s the cell up to: Quiescent, Activated, Apoptosing? Ref: Beckman Coulter Inc: http://www.beckman.com Dead Cells Viable Cells Early Apoptosis Late Apoptosis
  • The Pre-Apoptotic Sequence Ref: Backway KL. McCulloch EA. Chow S. Hedley DW. Cancer Research. 57 (12):2446-51, 1997
  • What’s its history?
    • Time course
    • BrdU
    • Topical staining
    • Tracking
  • What’s its history?
    • Time course
    • BrdU
    • Topical staining
    • Tracking
  • What’s its history?
    • Time course
    • BrdU
    • Topical staining
    • Tracking
    10 0 10 1 10 2 10 3 10 4 CFSE Fluorescence Cell divisions
  • And..
    • Cytotoxicity
    • Chromosomes
    • Parasites
    • ...
  • Instruments at WEHI
    • SORTERS
      • FACStar+
      • FACSVantageSEDiVa
      • MoFlo
      • FACSAria (2)
    • ANALYSERS
      • FACScan (2)
      • FACSCalibur (2)
      • LSR I
      • LSR II (2)
  • WEHI Instruments: Sorters
    • Run by FACS lab staff 10:15-17:00 working days only
    • Special arrangements over lunch and after 17:00
    • DIY operation after hours by trained, experienced users
  • 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_booking/lab/ ) .
    • Advance schedules are set by Monday morning lottery .
    • Time allocation is qualified-random (ask for details).
  • WEHI Intruments: Analysers
    • Do-It-Yourself analysis, available anytime.
    • Training provided as well as troubleshooting and data interpretation assistance on request.
  • 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.
  • 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.
  • 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 .
  • 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").
  • Cytometry (FACS) Advanced Technology Laboratory
    • FACS Team:
            • Frank
            • Sandy
            • Angela
            • David
            • Hesham
            • Rumbi
            • Dora
            • Padmini
            • Lankesha (on leave)
    http://www.wehi.edu.au/cytometry/presentations.html