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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. Structures on or within a cell are marked with fluorescent tags.

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Cytometry (FACS) Advanced Technology Laboratory

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  1. 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

  2. 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.

  3. Flow Cytometry: Multi-colour Detection

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

  5. Flow Cytometry and Sorting: Schematic

  6. Questions Answered by Flow Cytometry • What kind of cell? • What’s it up to? • What’s its history? • And..

  7. What Kind of Cell? • Immunofluorescence • Specific antigen • Genetic reporters • FACS Gal • FISH • Fluorescent Protein

  8. What Kind of Cell? • Immunofluorescence • Specific antigen • Genetic reporters • FACS Gal • FISH • Fluorescent Protein

  9. What Kind of Cell? • Immunofluorescence • Specific antigen • Genetic reporters • FACS Gal • FISH • Fluorescent Protein

  10. What Kind of Cell? • Immunofluorescence • Specific antigen • Genetic reporters • FACS Gal • FISH • Fluorescent Protein

  11. 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

  12. 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

  13. 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

  14. What’s its history? • Time course • BrdU • Topical staining • Tracking

  15. What’s its history? • Time course • BrdU • Topical staining • Tracking

  16. What’s its history? • Time course • BrdU • Topical staining • Tracking Cell divisions 100 101 102 103 104 CFSE Fluorescence

  17. And.. • Cytotoxicity • Chromosomes • Parasites • ...

  18. Instruments at WEHI SORTERS • FACStar+ • FACSVantageSEDiVa • MoFlo • FACSAria (2) ANALYSERS • FACScan (2) • FACSCalibur (2) • LSR I • LSR II (2)

  19. 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

  20. 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).

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

  22. 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.

  23. 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.

  24. 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 .

  25. 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").

  26. 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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