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Flow Cytometry at Boston University Medical Campus

Flow Cytometry at Boston University Medical Campus. Introduction to some methods that we offer. Yan Deng (X4-5225), ydeng@bu.edu. Gerald Denis (X4-1371), gdenis@bu.edu. Definitions. Flow cytometry simultaneously measures and analyzes

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Flow Cytometry at Boston University Medical Campus

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  1. Flow Cytometry at Boston University Medical Campus Introduction to some methods that we offer Yan Deng (X4-5225), ydeng@bu.edu Gerald Denis (X4-1371), gdenis@bu.edu

  2. Definitions Flow cytometry simultaneously measures and analyzes multiple physical characteristics of single particles, usually cells, as they move in a fluid stream through a beam of light. Any suspended particle between 0.2 and 50μM is suitable. Larger particles, solid tissue or clumps of cells must be disaggregated to be analyzed. Examples: lymphocytes, protozoa, micron beads, chromosomes

  3. Definitions The particles in the fluid stream scatter incident light, which reveals internal properties, size and granularity. The particles also fluoresce; they emit laser light at the interrogation point; this light is picked up by detectors arrayed at a different angle to detectors of scattered light.

  4. fluidics optics electronics

  5. Fluidics waste flow cell sheath fluid laser sample ~2 x 105 to 1 x 107 cells/ml

  6. Electronics digitize

  7. ● low flow rate ● high flow rate ● narrow sample core ● wide sample core ● high resolution ● low resolution sample fluid Fluidics sheath fluid • Purpose of the fluidics system: • Transport particles in a fluid stream • to the laser beam to be interrogated • 2. Position the sample core in the center • of the laser beam ‘hydrodynamic focusing’ single file particles

  8. Fluidics • Concerns • Shear rates for cells: check after you complete • a run to ensure that the cells are intact. • 2. Larger tips are needed for cell sorting.

  9. The laser beam is focused on the sample core; lasers must be fixed in place. Excitation is accomplished by lasers that emit light at specified wavelengths. The atomic properties of the excitation media define this wavelength for a particular laser. Argon blue lasers emit light at 488 nm. This is the most common and versatile wavelength for excitation of fluorochromes. Optics

  10. Fluorescein (FITC) Propidium iodide (PI) Hoechst 33258 Texas Red Laser light must overlap with excitation wavelength

  11. Filters resolve overlapping wavelengths of emitted light Longpass filter: transmits light of longer than or equal to a specific wavelength Shortpass filter: transmits light of shorter than or equal to a specific wavelength Bandpass filter: transmits light only within a narrow range of wavelengths Optics

  12. Excitation l of Texas Red is not optimal with 488 nm laser

  13. http://probes.invitrogen.com/resources/spectraviewer/ http://www.bdbiosciences.com/spectra/ For more information

  14. Electronics The electronic system: 1. quantifies the voltage pulse 2. converts analog signals to digital values 3. performs compensation 4. transfers data to the computer for analysis.

  15. Adjusting the voltage of the PMT helps to optimize capture of desired populations Electronics – Photomultiplier Tubes (PMTs)

  16. DNA content (Linear detection) DNA content (Log detection) Electronics - Amplifiers Amplifiers are of two types: linear or logarithmic Linear amplification is typically used with scatter. Logarithmic amplification is typically used with fluorescence.

  17. Electronics Threshold is the minimum pulse height above which a signal will be processed electronically.

  18. before after Electronics When a threshold value is defined, only signals with intensities greater than or equal to the value are recorded. Adjustment of blood immunophenotype to exclude debris

  19. Fluorescence

  20. Fluorescence Fluorescence intensity is proportional to the number of binding sites of the fluorescent compound, or “fluorochrome”. Remember, when labeling live cells, to keep the experiment at 40C, because receptors are internalized and recycled, leaving their fluorochromes behind.

  21. positive stain negative stain Immunophenotyping

  22. Cluster of Differentiation antibody specificity CD3 Pan-T lymphocytes CD4 T helper/Inducer CD5 T cells, subset B cells CD11b Monocytes, granulocytes, NK/T cells CD19 B cells CD20 B cells CD25 Il-2R, Tac IL-2 receptor, Activated T cells, B cells, NK cells, monocytes CD34 Progenitor cells CD69 Early activation antigen on T, B and NK cells

  23. 44% 1% CD3-PE 48% 7% CD19-FITC Immunophenotyping Two-color fluorescence of lymphocytes Note the experiment-specific definition of the “negative” population

  24. Spectral overlap Fluorescence Multicolor analysis

  25. Uncompensated Optimal Compensation To correct for emission spillover of FITC signal (normally detected in the FL1 channel) into the FL2 channel (which detects PE), it is necessary to use filters or electronic compensation or both.

  26. Before After Compensation Multicolor immunophenotyping

  27. Data presentation formats

  28. Gates

  29. Gates

  30. The uses of gates for cell sorting

  31. Cell cycle kinetics Multiparameter sorting Some applications

  32. DNA content for cell cycle / apoptosis Immune system activation Some applications

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