1. www.le.ac.uk Flow Cytometry

2. Applications FRET- protein interaction Membrane protein expression Intracellular protein expression Cell viability Ca 2+ flux Cell cycle analysis Cell sorting (FACs) Intracellular pH RNA content Cell proliferation Production of intracellular oxidative species

3. General principles Fluidics Optics Detection Output

4. Fluidics Sample uptake –Older machines under vacuum –Newer machines inject sample –Under high pressure Injected into centre of a flow chamber Buffer or dH2O (Sheath fluid) flow round the sample causing cells to form single line –Hydrodynamic focusing

5. Optics Samples move through path of laser as single cells –Different types of laser Violet laser – 405nm Blue laser – 488nm Yellow laser – 561nm Red laser – 635nm –Numbers represent wavelengths at which the lasers excite Fluorophore's –The blue laser is also used to measure size (forward scatter) and granularity of cells (side scatter)

6. Optics continued… Laser light reflects off cells and reaches dichroic mirrors –Mirrors allow certain wavelengths through and all other wavelength's are reflected to detectors –There are three types of filter Long pass – allows light through over a certain wavelength –E.g. 750LP- light >750 Short pass – allows light through below a certain wavelength –E.g. 560SP- light <560nm Bandpass – allows light through between certain wavelengths –E.g. 530/20 – light between 520 and 540nm The fluorophore’s you are able to use are dependent on the filters you have – and their emission wavelength

7. Optics continued…

8. Detection Detected light hits photomultiplier tubes (PMTs) Creates voltage pulse proportional to amount of light they receive –E.g. small cell, low forward scatter, small pulse –Large cell, high forward scatter, large pulse Voltages from fluorescent signals converted to numerical values

9. Output Data collected represented in graphical format Histogram, dot plot, contours…

10. Important considerations Fluorophore’s / Lasers Location of molecule of interest Controls –Isotype –Secondary control –Positive Compensation Gating Method

11. Fluorophore’s APC-H7 APC-Cy7 APC-Vio770 APC PerCP-Cy5.5 PerCP PE-Vio770 PE-Cy7 PE-Cy5.5 PE-Cy5 PE-Texas Red PE FITC Pacific Orange VioGreen AmCyan Pacific Blue VioBlue PI Alexa Fluors’s Lots more

12. Fluorophore’s continued… The Fluorophore's you can use depend on your lasers and filters E.g. FITC (green) Excited 495nm, emission 519nm –Stokes shift –Use 488 blue laser and 530/40 band pass filter

13. Fluorophore’s

14. Controls-1 Isotype –Antibody from the same species as host and same antibody isotype E.g. we have a mouse IgG2a anti-CD14-FITC antibody so an isotype would be mouse IgG2a-FITC, usually used at same concentration under same conditions Allow a measure of non-specific binding

16. Controls-2 Sometimes primary antibody to molecule of interest isn’t labelled Have to use labelled secondary antibody to detect primary Standard procedure to use a secondary only sample as control

17. Controls-3 Positive controls –Need to know if antibody works –Optimum concentration of primary antibody –Best method to stain

18. Compensation If using more than one fluorophore Needed if Fluorophore's excited by same laser Fluorophore’s have emission spectra –Values given are peak emission values In the real world emission spectra are graded

19. Compensation continued… Some emission spectra of fluorophore’s overlap Means that they will fluoresce in that channel If no compensation than you would not know if your result is real Most new machines now have automatic settings

20. Where is your molecule of interest? Membrane –Staining relatively simple no fixation or permeabilisation required Cytosol/organelle –Permeabilisation and fixation required Protein? DNA? Phospholipid? Antibodies need to be able to enter a cell Some dyes don’t e.g. Calcein

21. Methods Each lab has standard methods Usually results do not differ much between similar methods However, different methods need to be used if protein intra/extracellular –Permeabilisation Acetone / Tween / Triton –Fixation Methanol / PFA / Formaldehyde Also need to lyse RBCs if using whole blood

22. Example Protein expressed on surface of endothelial cells Cultured Ea.Hy926 cells –Know contain protein by western blotting

23. Conventional method Resuspend cells in buffer Stain 30min primary antibody Stain 30min secondary antibody Make up to 1ml in buffer Run on flow cytometer

24. Adjusted method Cells fixed for 5min in methanol Resuspended in buffer Stained as before

25. Gating Gates can be used: –To select populations –To measure the level of protein expression E.g. in whole blood can gate monocytes based on size and granularity Granulocytes Monocytes Lymphocytes

26. Gating continued… Can set histogram to identify molecule of interest on gated cells

27. Gating continued… When measuring level of protein expression etc. the isotype is usually set to 2% To take into account cells that express the molecule of interest weakly

28. Stats Can get a number of statistics from the data Most useful –Percentage of positive cells –Median fluorescence Intensity and spread of distribution

29. Worked example

30. PE

31. FITC

32. Compensation

34. Isotype 0h 4h 8h

35. Facilities in the department Currently 2 flow cytometers Both Beckman Coulter –Gallios –ADP Cyan MUST BE TRAINED AND HAVE INDUCTION FROM TINA JAMES BEFORE YOU START USING EITHER MACHINE!!

36. Gallios 3 lasers, 488 (blue), 638 (red), 405 (violet) Filters allow up 10 colours –FL1 525/40 bp –FL2 575/30 bp –FL3 620/30 bp –FL4 695/30 bp –FL5 755 LP lp –FL6 450/40 bp –FL7 550/40 bp –FL8 660/20 bp –FL9 725/20 bp –FL10 755 lp Blue Violet Red Brand new Carousel so can load and leave Kaluza software, easy to use

37. ADP Cyan 3 lasers, 488 (blue), 405 (violet) and 635 (red) –Although red filter temperamental and not always aligned Filters allow up 9 colours –FL1 530/40 bp –FL2 575/25 bp –FL3 613/20 bp –FL4 680/30 bp –FL5 750 LP –FL6 450/50 bp –FL7 530/40 bp –FL8 665/20 bp –FL9750 LP Blue Violet Red No carousel so have to manually load samples Software easy to use and transferable to work computer Can add gates, analyse and set up histograms after running samples

38. Documentation Keeney, M., et al., Isotype controls in the analysis of lymphocytes and CD34+ stem and progenitor cells by flow cytometry - Time to let go! Cytometry, 1998. 34(6): p. 280-283. http://www.beckmancoulter.com/wsrportal/wsr/research-and- discovery/products-and-services/flow-cytometry/flow-cytometers/cyan- adp-analyzer/index.htm - For CYAN ADP guide http://www.biolegend.com/spectraanalyzer Flow Cytometry- a basic introduction, Michael G. Ormerod Most of the companies have information on flow cytometry I also have 4/5 power points and training manuals from Beckman Coulter