1. Fundamentals and Applications of Flow Cytometry Scott Tighe Flow Cytometry Core Lab at the Vermont Cancer Center HSRF 305 656-2557

2. Overview Basics components of a cytometer Fundamentals of photonics-optics and fluorescence Software and modeling Types of analysis Sorting for cells and RNA Sample requirements- [controls, compensation, titer Ab, blocking] Sign-up for time on VCC instruments

3. What is Flow Cytometry? An instrument for making cell-based fluorescent measurements. Method for quantitating cellular or structural components of a cell using fluorescent antibodies or probes. Allows Analysis of tens of thousands of cells in minutes. A method to sort and collect specific cell types. The BD FACS ARIA with sorter Beckman Coulter Epics XL

4. Hardware Components of Flow Cytometer Fluidics - Cells are carried to laser in a saline-based sheath fluid. Pneumatics- Pressure drives the fluid flow Optics - laser, band-pass filters, and PMT detectors. Computer- Performs the analysis Older MoFlo cytometer by Cytomation

5. Fluidics Sheath Tank Waste Tank Line Pressure Vacuum Sample Pressure Sheath Pressure (Constant) THE FLOW CELL Laser focusing and hydrodynamic focusing LASER

6. Injector Tip Fluorescence signals Focused laser beam Sheath fluid The Flow cell: showing hydrodynamic focusing Higher the sample flow the wider the sample stream and lower the resolution

7. Excitation Sources

8. Lets consider two items The electromagnetic spectrum Fluorochrome excitation-emission curve

9. Lasers provide Coherent Light (Single mode-single wavelength) High Power Narrow band width Can be tunable or fixed wavelength Gas tube or Pumped solid state-which allows coverage for all fluorochromes! GASPSS Krypton-647nmNd-Yag (AlGaAs diode or Krypton lamp pumped) Argon -488nm Freq. Tripling !!! 1064,532,355nm HeNe [633nm]Great for Flow cytometry UV-364nm [from Ar]Ti:sapphire (Ar pumped) 650-1100 nm Excimers [193-284nm] Lasers This is just a few….

10. Output frequencies of common tunable gas tube lasers

11. Light Emitting Diodes [LED] -Benefits –Cheaper –Smaller –Easily Available –Many wavelengths -Cons -Not as bright -Broad excitation spectra -Not available for UV

12. Fluorescence and Photons

13. Fluorescence the molecular absorption of a photon triggers the emission of another photon with a longer wavelength. Release of Photon Stoke shift Ext. coef + Quantum yield is Quantum efficiency

14. The fluorochrome: Phycoerytherin Excitation 488 nm Emission Maximum 575 nm >20 chromophores per molecule High quantum yield (bright) 488 nm Argon 575 nm Large multi-subunit, globular (~240 kDa)

16. Optics and Detection

17. Laser (Argon 488nm) Red Signal FS Sensor Fluorescence detector (PMT1, PMT4 etc.) Detecting and sorting of different colors from cellular emission involves band pass filter and beam splitters Green Signal

18. Optics in a Flow Cytometer Beam Splitters

19. FS Sensor [size] Laser Forward Angle [FS] Light Scatter Provides data on size- The bigger the cell, the larger the FS

20. Side Scatter [SS] Detector Provides data on internal structures The more structures, the more ss Granulocytes have high ss Provides data on surface characteristics Dead cell have a rougher cell surface and a higher ss RBC have little to no ss FS Sensor SS Sensor [granularity] Laser

21. Beam Splitters Dichroic Filter/Mirror at 45 degrees Reflected light Transmitted Light Laser Light+ Sample Light 500-680nm BS 625 625nm and above 500-624 reflected 488-680nm 500LP

22. Band Pass Filters Transmitted Light White Light Source 630/30nm BandPass Filter A 630/30 BPF will only allow 615-645nm through

23. Long Pass Filters Light Source 520 nm Long Pass Filter >520 nm Light Light Source 575 nm Short Pass Filter <575 nm Light Short Pass Filters Short Pass Filters Note: Great for microscopes

24. Detectors

25. Photomultiplier tubes (PMT) “Old” but good technology Most common detector used in flow High sensitivity but poor quantum efficiencies in red (>650nm) Able to adjust gain to over 100,000 Common Inexpensive Photodiode (PD) New[er] technology, still not common High quantum efficiencies for visible No internal gain adjustment Requires Cooling Require a high voltage bias

26. Flow cytometers may have 4 to 6 detector channels [or more] Flow cell PMT=Photomultiplier Tubes-do not see colors, only photons. PMT Dichroic filters Bandpass filter Laser 1 2 3 4 PMT

27. Spectral Compensation Must be performed when using fluorochromes with overlapping emissions

28. Sorting Cells

29. The Cytomation Mo Flo cell sorter Analyzes and sorts cells at 70,000 cells per second Cost $ 350,000 

30. 488 nm laser + - Charged Plates Single cells sorted into test tubes FS Sensor Fluorescence detector Sorting

31. Data Output

32. Histograms Data Output is represented by a histogram -Single parameter trace [one color] -Dual paramater dot plot [two color] Fluorescent intensity- 

33. Gating Allows the ability to select specific cell populations in one histogram and analyze for additional parameters [colors] in additional histograms

34. Comparison of single and dual parameter histograms

35. Specific Types of Analysis Done Using Flow Cytometry

36. Some Typical Applications of Flow Cytometry ? Immunophenotyping DNA cell cycle/tumor ploidy Membrane potential Ion flux Cell viability Karyotyping Cell tracking and proliferation Sorting Redox state Chromatin structure Cell proliferation assay Cell enumeration and sizing Apoptosis Phagocytosis Intracellular pH Intracellular calcium Oxidative burst Intracellular antigen measurement Cytokine detection Reticulocyte analysis Platelet analysis

37. Immunophenotyping

38. Immunophenotyping Classifying immune cells using cell surface antigens CD3 CD4 CD # = cluster designation number CD3-T-cell CD4-T helper

39. The structure of IgG-An Antibody Fab regions Fc receptor

40. Two Types of Antibody Labeling Direct labeling : Uses one antibody that has a fluorochrome conjugated directly on it. One step staining. Easier. Cell Indirect labeling : Uses two antibodies.The first is “against” a specific antigen on the cell. The second antibody is fluorochrome-labeled and is “against” the first. More complicated. Cell

41. Example of Data Using two mABs with dyes of different color outputs.1 1 10 1001000

42. Non-Specific Antigen Blocking Blocking is important to avoid false positives Non-specific binding of antibodies is really Fc binding Typically a serum source (BSA, FCS) is used but is often not adequate. Recommend goat IgG at 100-200ug/ml

43. mAB Titering-A must!!!! Uses a specific Number of cells against antibody dilutions. Perform on new lots of antibodies

44. Cell Cycle

45. Cell Cycle Analysis Indicates the rate and stage of cell replication or division. Propidium Iodide most common dye. The dye intercalates into the DNA strand. G2G2G2G2 M G0G0G0G0 G1G1G1G1 s CountCount 0 200 400 600 8001000 2NG1/G0 4NG2M S Phase (Synthesis)

46. Modeling Cell Cycle data When peaks are close together and overlapping, it is important to use specific software to model the data and get accurate results ModFit 3D 3.0 WinList 5.0 FloJo WinMDI is a free software written by J.Trotter and is available on the web.

47. Cell Cycle of GFP Cells

48. Cell Viability

49. Cell Viability Simplest method using cell permeabilization -Propidium Iodide -7AAD -Sytox Membrane potentials are a good indicator such mitochondria membrane potential [JC1]. Redox dyes-DHF, DHR- Turn colorless by reduction in cells. Enzyme activity probes-Esterase activity using cFDA, cell tracker dyes, calcein,… converted to fluorescent probe by enzymes.

50. How the assay works: PI cannot normally cross the cell membrane If the PI penetrates the cell membrane, it is assumed to be damaged Cells that are brightly fluorescent with the PI are damaged or dead PI PI PI PI PI PI PI PI PI PI PI PI PI PI Cell Viability using Dye Exclusion-Propidium Iodide Pi fluoresence >>> Dead Live

51. Apoptosis

52. Apoptosis-Annexin staining Uses Annexin-FITC against Phosphatidylserine to determine membrane translocation Uses PI to determine membrane permeability Must be used cautiously on adherent cells do to trypsin Not for fixed cells PI Annexin ApoptoticNecrotic

53. APO-BRDU-TUNEL DNA fragmentation Available 3’OH Uses Tdt to add BrDU Stain with anti-BrDU

54. Proliferation Assays

55. Proliferation can be measured by cell cycle, Brdu incorporation, and membrane dyes such as CFSE and PKH26. CFSE, PKH 26, Brdu incorporation to S-phase DNA

56. Sorting cells for RNA Decon the flow cytometer with bleach Run a test on dummy cells first check your viability Ensure all reagents are RNase-free Sort into an extraction buffer when applicable or sort into sterile media Extract RNA on the same day if in an extraction buffer *****Please come get a handout at our lab regarding these protocols

57. Sample requirements Negative no stain controls are required for most runs Single color controls are required for spectral compensation when performing two or more color analysis Samples should be in 12x75 plastic tubes containing 800ul of cell suspension at a concentration of 100,000-1 million cells Cells that are clumpy must be filter thru 70um mesh If using dual antibody staining, a secondary only will be necessary as well as your no stain control All analysis types should be accompanied with a positive control sample in order to validate the staining, protocol, and compensation

58. Logging into the VCC Flow cytometer Billing and sign up is done using the BioDesktop DNA facility>>>Shared Inst. Sign-up>>>Flow cytometer Reserve the time you plan to use it >>>OK Enter your contact info Enter your chartstrings into the chartstring manager You may edit after your run An email will also be sent to you in case you need to edit your time for billing purposes Please sign up in the log-in book as well The new rate for the VCC flow is $9.51 [cancer] and 19.01 [non-cancer] per ½ blocks

59. Thank you for your time