1. Cytometry (FACS) Advanced Technology Laboratory FACS Team: Frank Frank Viki Viki Sandy Sandy Padmini (on leave) Padmini (on leave) Sophie Sophie Lankesha Lankesha Angela Angela Dora Dora David 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. Structures on or within a cell are marked with fluorescent tags. Single cells suspended in saline are streamed past a focused laser beam. Single cells suspended in saline are streamed past a focused laser 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 processed and interpreted by computer. 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). FSC detectors correlate with cell size (cross-sectional area, refractive index). SSC detectors indicates nuclear shape and cytoplasmic granularity. 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 kind of cell? What’s it up to? What’s it up to? What’s its history? What’s its history? And.. And..

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

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

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

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

11. What’s the cell up to: Quiescent, Activated, Apoptosing? Total DNA: Cell Cycle Total DNA: Cell Cycle RNA Synthesis RNA Synthesis Mitochondria Mitochondria Intra-cellular Ca ++ Intra-cellular Ca ++ Intra-cellular pH Intra-cellular pH Phosphatidyl Serine Exposure Phosphatidyl Serine Exposure

12. What’s the cell up to: Quiescent, Activated, Apoptosing? Total DNA: Cell Cycle Total DNA: Cell Cycle RNA Synthesis RNA Synthesis Mitochondria Mitochondria Intra-cellular Ca ++ Intra-cellular Ca ++ Intra-cellular pH Intra-cellular pH Phosphatidyl Serine Exposure Phosphatidyl Serine Exposure G0/1 S G2/M

13. What’s the cell up to: Quiescent, Activated, Apoptosing? Total DNA: Cell Cycle Total DNA: Cell Cycle RNA Synthesis RNA Synthesis Mitochondria Mitochondria Intra-cellular Ca ++ Intra-cellular Ca ++ Intra-cellular pH Intra-cellular pH Phosphatidyl Serine Exposure Phosphatidyl Serine Exposure

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

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

16. What’s its history? Time course Time course BrdU BrdU Topical staining Topical staining Tracking Tracking 10 0 10 1 10 2 10 3 10 4 CFSE Fluorescence Cell divisions

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

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

19. WEHI Instruments: Sorters Run by FACS lab staff 10:15-17:00 working days only Run by FACS lab staff 10:15-17:00 working days only Special arrangements over lunch and after 17:00 Special arrangements over lunch and after 17:00 DIY operation after hours by trained, experienced users 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. 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/). 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/).http://unix44.alpha.wehi.edu.au/facs_booking/lab/ Advance schedules are set by Monday morning lottery. Advance schedules are set by Monday morning lottery. Time allocation is qualified-random (ask for details). Time allocation is qualified-random (ask for details).

21. WEHI Intruments: Analysers Do-It-Yourself analysis, available anytime. Do-It-Yourself analysis, available anytime. Training provided as well as troubleshooting and data interpretation assistance on request. 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 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) 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. 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. 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. 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/). Go online up to 3 weeks in advance (http://unix44.alpha.wehi.edu.au/facs_booking/user/). Booking is instantaneous. Booking is instantaneous. Cancellations more than 24 hrs in advance attract no penalty. 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. 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 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. 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. 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. 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. 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"). FACS analysers must be decontaminated after use to avoid cross contamination (see "rules of operation").

26. Cytometry (FACS) Advanced Technology Laboratory FACS Team: Frank Frank Viki Viki Sandy Sandy Padmini (on leave) Padmini (on leave) Sophie Sophie Lankesha Lankesha Angela Angela Dora Dora David David http://www.wehi.edu.au/cytometry/presentations.html