Cytometry (FACS)Cytometry (FACS)Advanced Technology LaboratoryAdvanced Technology Laboratory

FACS Team: FACS Team: FrankFrankVikiVikiSandySandyPadmini (on leave)Padmini (on leave)SophieSophieLankeshaLankeshaAngelaAngelaDoraDoraDavidDavid

http://www.wehi.edu.au/cytometry/presentations.htmlhttp://www.wehi.edu.au/cytometry/presentations.html

How Flow Cytometry WorksHow Flow Cytometry Works

Structures on or within a cell are marked with fluorescent tags. Structures on or within a cell are marked with fluorescent tags. Single cells suspended in saline are streamed past a focused laser Single cells suspended in saline are streamed past a focused laser

beam.beam. Laser light excites fluorescence and scatters in all directions.Laser light excites fluorescence and scatters in all directions. Detectors receive flashes of fluorescent and scattered light that are Detectors receive flashes of fluorescent and scattered light that are

processed and interpreted by computer. processed and interpreted by computer.

Flow Flow Cytometry: Cytometry: Multi-colour Multi-colour DetectionDetection

Scattered Light InterpretationScattered Light Interpretation

FSC detectors correlate with cell size FSC detectors correlate with cell size (cross-sectional area, refractive index).(cross-sectional area, refractive index).

SSC detectors indicates nuclear shape SSC detectors indicates nuclear shape and cytoplasmic granularity. and cytoplasmic granularity.

Flow Cytometry and Sorting: Flow Cytometry and Sorting: SchematicSchematic

Questions Answered by Flow Questions Answered by Flow CytometryCytometry

What kind of cell?What kind of cell? What’s it up to?What’s it up to? What’s its history?What’s its history? And..And..

What Kind of Cell?What Kind of Cell?

ImmunofluorescenceImmunofluorescence Specific antigenSpecific antigen Genetic reportersGenetic reporters

FACS GalFACS Gal FISHFISH Fluorescent ProteinFluorescent Protein

What Kind of Cell?What Kind of Cell?

ImmunofluorescenceImmunofluorescence Specific antigenSpecific antigen Genetic reportersGenetic reporters

FACS GalFACS Gal FISHFISH Fluorescent ProteinFluorescent Protein

What Kind of Cell?What Kind of Cell?

ImmunofluorescenceImmunofluorescence Specific antigenSpecific antigen Genetic reportersGenetic reporters

FACS GalFACS Gal FISHFISH Fluorescent ProteinFluorescent Protein

What Kind of Cell?What Kind of Cell?

ImmunofluorescenceImmunofluorescence Specific antigenSpecific antigen Genetic reportersGenetic reporters

FACS GalFACS Gal FISHFISH Fluorescent ProteinFluorescent Protein

What’s the cell up to: Quiescent, What’s the cell up to: Quiescent, Activated, Apoptosing?Activated, Apoptosing?

Total DNA: Cell CycleTotal DNA: Cell Cycle RNA SynthesisRNA Synthesis MitochondriaMitochondria Intra-cellular CaIntra-cellular Ca++++

Intra-cellular pHIntra-cellular pH Phosphatidyl Serine ExposurePhosphatidyl Serine Exposure

What’s the cell up to: Quiescent, What’s the cell up to: Quiescent, Activated, Apoptosing?Activated, Apoptosing?

Total DNA: Cell CycleTotal DNA: Cell Cycle RNA SynthesisRNA Synthesis MitochondriaMitochondria Intra-cellular CaIntra-cellular Ca++++

Intra-cellular pHIntra-cellular pH Phosphatidyl Serine ExposurePhosphatidyl Serine Exposure

G0/1

SG2/M

What’s the cell up to: Quiescent, What’s the cell up to: Quiescent, Activated, Apoptosing?Activated, Apoptosing?

Total DNA: Cell CycleTotal DNA: Cell Cycle RNA SynthesisRNA Synthesis MitochondriaMitochondria Intra-cellular CaIntra-cellular Ca++++

Intra-cellular pHIntra-cellular pH Phosphatidyl Serine ExposurePhosphatidyl Serine Exposure

What’s its history?What’s its history?

Time courseTime course BrdUBrdU Topical stainingTopical staining TrackingTracking

What’s its history?What’s its history?

Time courseTime course BrdUBrdU Topical stainingTopical staining TrackingTracking

What’s its history?What’s its history?

Time courseTime course BrdUBrdU Topical stainingTopical staining TrackingTracking

100 101 102 103 104

CFSE Fluorescence

Cell divisions

And..And..

CytotoxicityCytotoxicity ChromosomesChromosomes ParasitesParasites ......

Instruments at WEHIInstruments at WEHISORTERSSORTERS

FACStar+FACStar+ FACSVantageSEDiVaFACSVantageSEDiVa MoFloMoFlo FACSAria (2)FACSAria (2)

ANALYSERSANALYSERS FACScan (2)FACScan (2) FACSCalibur (2)FACSCalibur (2) LSR ILSR I LSR II (2)LSR II (2)

WEHI Instruments: SortersWEHI Instruments: Sorters Run by FACS lab staff 10:15-17:00 working Run by FACS lab staff 10:15-17:00 working

days only days only Special arrangements over lunch and after Special arrangements over lunch and after

17:0017:00 DIY operation after hours by trained, DIY operation after hours by trained,

experienced usersexperienced users

Booking of Time: SortersBooking of Time: Sorters

Consult lab staff or go onlineConsult lab staff or go online to discover to discover suitable vacancies already on the schedule. suitable vacancies already on the schedule. Requests are vetted by lab staff. Requests are vetted by lab staff.

If there are no current vacancies, If there are no current vacancies, fill in a fill in a paper form or go onlinepaper form or go online to request time 3 to request time 3 weeks in advance weeks in advance ((http://unix44.alpha.http://unix44.alpha.wehiwehi.edu.au/.edu.au/facsfacs_booking/lab/_booking/lab/))..

Advance schedules are set by Monday Advance schedules are set by Monday morning morning lotterylottery. .

Time allocation is Time allocation is qualified-randomqualified-random (ask for (ask for details). details).

WEHI Intruments: AnalysersWEHI Intruments: Analysers

Do-It-Yourself analysis, available Do-It-Yourself analysis, available anytime. anytime.

Training provided as well as Training provided as well as troubleshooting and data interpretation troubleshooting and data interpretation assistance on request. assistance on request.

Training: DIY Flow CytometryTraining: DIY Flow Cytometry

Step 1: Multi-media interactive computerised training Step 1: Multi-media interactive computerised training for basic cytometry which is advised also for those for basic cytometry which is advised also for those planning cell sorter use. (enquire at FACS Lab). planning cell sorter use. (enquire at FACS Lab).

Step 2: Group tutorials offered from time to time (look Step 2: Group tutorials offered from time to time (look for intranet notices)for intranet notices)

Thereafter, individual supervision (by your supervisor Thereafter, individual supervision (by your supervisor or by FACS lab staff) with your first and subsequent or by FACS lab staff) with your first and subsequent flow cytometric analyses should be sought. flow cytometric analyses should be sought.

Prospective LSR II users are advised to become Prospective LSR II users are advised to become familiar first with FACScan/FACSCalibur operation. familiar first with FACScan/FACSCalibur operation.

Data analysis demonstrations and advice can be Data analysis demonstrations and advice can be sought at any time. sought at any time.

Booking of Time: AnalysersBooking of Time: Analysers

Go onlineGo online up to 3 weeks in advance up to 3 weeks in advance (http://(http://unix44.alpha.wehi.edu.au/facs_booking/user/)unix44.alpha.wehi.edu.au/facs_booking/user/)..

Booking is instantaneous. Booking is instantaneous. Cancellations more than 24 hrs in advance Cancellations more than 24 hrs in advance

attract no penalty. attract no penalty.

FACS Data AnalysisFACS Data Analysis Stored FACS data from sorters and analysers Stored FACS data from sorters and analysers

may be analysed and displayed on PC or may be analysed and displayed on PC or Macintosh using WEHI's "Macintosh using WEHI's "WEASELWEASEL" program. " program.

Specialist DNA analyses are performed using Specialist DNA analyses are performed using ""ModFitLTModFitLT" one copy of which is available at " one copy of which is available at WEHI. WEHI.

Specialist cluster analysis is performed using Specialist cluster analysis is performed using ""ACluEACluE", available on request for PC or ", available on request for PC or Macintosh . Macintosh .

Bio-safetyBio-safety Cell sorters produce Cell sorters produce aerosolsaerosols so identification of so identification of

biohazardous samples is compulsory prior to biohazardous samples is compulsory prior to booking. booking.

Biosafety committee approvalBiosafety committee approval is required before is required before initiating projects involving initiating projects involving analysisanalysis or or sortingsorting of of samples with a known or potential biohazard, samples with a known or potential biohazard, including all un-fixed human cells.including all un-fixed human cells.

Human samplesHuman samples will be sorted only on selected cell will be sorted only on selected cell sorters in the Cytometry Lab. sorters in the Cytometry Lab.

FACS analysers must be FACS analysers must be decontaminateddecontaminated after use after use to avoid cross contamination (see "rules of to avoid cross contamination (see "rules of operation"). operation").

Cytometry (FACS)Cytometry (FACS)Advanced Technology LaboratoryAdvanced Technology Laboratory

FACS Team: FACS Team: FrankFrankVikiVikiSandySandyPadmini (on leave)Padmini (on leave)SophieSophieLankeshaLankeshaAngelaAngelaDoraDoraDavidDavid

http://www.wehi.edu.au/cytometry/presentations.htmlhttp://www.wehi.edu.au/cytometry/presentations.html