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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

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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.


Flow cytometry multi colour detection

Flow Cytometry: Multi-colour Detection


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.


Flow cytometry and sorting schematic

Flow Cytometry and Sorting: Schematic


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


Cytometry facs advanced technology laboratory

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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