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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.html. How Flow Cytometry Works. Structures on or within a cell are marked with fluorescent tags.

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cytometry facs advanced technology laboratory

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.html

how flow cytometry works
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.
scattered light interpretation
Scattered Light Interpretation
  • FSC detectors correlate with cell size (cross-sectional area, refractive index).
  • SSC detectors indicates nuclear shape and cytoplasmic granularity.
questions answered by flow cytometry
Questions Answered by Flow Cytometry
  • What kind of cell?
  • What’s it up to?
  • What’s its history?
  • And..
what kind of cell
What Kind of Cell?
  • Immunofluorescence
  • Specific antigen
  • Genetic reporters
    • FACS Gal
    • FISH
    • Fluorescent Protein
what kind of cell1
What Kind of Cell?
  • Immunofluorescence
  • Specific antigen
  • Genetic reporters
    • FACS Gal
    • FISH
    • Fluorescent Protein
what kind of cell2
What Kind of Cell?
  • Immunofluorescence
  • Specific antigen
  • Genetic reporters
    • FACS Gal
    • FISH
    • Fluorescent Protein
what kind of cell3
What Kind of Cell?
  • Immunofluorescence
  • Specific antigen
  • Genetic reporters
    • FACS Gal
    • FISH
    • Fluorescent Protein
what s the cell up to quiescent activated apoptosing
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 apoptosing1
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

G2/M

S

what s the cell up to quiescent activated apoptosing2
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 its history
What’s its history?
  • Time course
  • BrdU
  • Topical staining
  • Tracking
what s its history1
What’s its history?
  • Time course
  • BrdU
  • Topical staining
  • Tracking
what s its history2
What’s its history?
  • Time course
  • BrdU
  • Topical staining
  • Tracking

Cell divisions

100

101

102

103

104

CFSE Fluorescence

slide17
And..
  • Cytotoxicity
  • Chromosomes
  • Parasites
  • ...
instruments at wehi
Instruments at WEHI

SORTERS

  • FACStar+
  • FACSVantageSEDiVa
  • MoFlo
  • FACSAria (2)

ANALYSERS

  • FACScan (2)
  • FACSCalibur (2)
  • LSR I
  • LSR II (2)
wehi instruments sorters
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
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
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
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
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
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
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 laboratory1

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.html

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