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What is Flow Cytometry ?

What is Flow Cytometry ?. Flow Cytometry. Introduction to Flow Cytometry. IGC Workshop. uic. Cell Sorting. Telma Lopes. IGC – Nov 12, 2009. Monitoring Sort. No Pain. No Sort!. Outline. Cell Sorters Drop Formation , Charging , and Deflection . Drop Delay . Sort-Drop Decisions

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What is Flow Cytometry ?

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  1. WhatisFlowCytometry? Flow Cytometry Introduction to Flow Cytometry IGC Workshop uic • CellSorting Telma Lopes IGC – Nov 12, 2009

  2. Monitoring Sort No Pain No Sort!

  3. Outline • Cell Sorters • DropFormation, Charging, andDeflection. • DropDelay. • Sort-DropDecisions • PurityandRecovery • Sample preparation • Applications

  4. CellSorters FACSAria • 100,000 eventspersecond • Blue, Red, Violet lasers • 9 simultaneouscolors • Cuvetteflowcell • FixedAlignment MoFlo • 100,000 eventspersecond • Blue, Red, UV, Yellow, Green lasers (only 3 atthetime) • 8 simultaneouscolors • Streaminair • Lasers alignedeverytimemachineisswitchedon

  5. Flow Sorting Process that allows the physical separation of a cell or particle of interest from a heterogeneous population • Droplet sorting: • Droplet generation - Break the stream into droplets • Charging the drop that contain the cell of interest • Defleting the charged drop A cell sorter sorts drops, not cells!

  6. DropFormation, Charging, andDeflection • First you need to know what you want to sort Flowcell Charge Collection Tubes Ifcellofinterestiswithinthe gate attimeof laser interrogationsorterwaitsuntilcellreachesthelastdrop (breakoffpoint) andchargesthedrop. Aspirator Tube • Sheath fluid must be a conductive ionic solution

  7. DropDelay Flow Cell cuvette At the moment of cells analysis (interrogation point) the cell is not in a drop Stream is subject to a vibration with a certain frequency Formation of drops stabilized Distan between the interrogation point and the breakoff point constant Nozzle All decisions are made on a theoretical drop at the interrogation point. Drop Delay Drop Breakoff From the drop delay information the system can calculate the position where the cell will be at the moment of droplet breakoff

  8. Expectation of Sorting • The aim is: • Sort ALL the cells of interest in the sample without losing one - Recovery • Sort ONLY the cells we want without sorting the wrong ones – Purity • Sort FAST without spending too much time in front of the instrument - Speed

  9. Reality Flow Cell Sorting relies on some statistical rules for randomly distributed events: Poisson Statistics cuvette Nozzle DropDelay Drop Breakoff These rules provide the theoretical background for calculating the achievable sort rates and the effectiveness of sorting

  10. Reality Flow Cell cuvette Nozzle DropDelay Cellsslightlyappartattheinterrogationpointmayendupinthesamedrop. Numberofevents/seccannotexceed 1/4 ofnumberofdrops/sec Drop Breakoff Ifsortinginpuritymode, sorterwillabortsortdecisioncompromisingrecovery. 1 cell for every 4 drops (ensuresthattheprobabilityofhaving more than 1 cellin a dropisalmost zero)

  11. Sort-DropDecisions IfsortinginPuritymode: • Decisions will compromise recovery A sort decision must be made for each particle. The sort decision is based on the sort gates AND sortmodes IfsortinginRecoverymode: • Decisions will compromise purity If sorting in Single-Cell mode: • High count accuracy

  12. PurityandRecovery Purity and recovery of the sort are mainly dependent on the sample 1. Quality of staining 2. Concentration 3. Adhesion properties of the cells (cell-cell, cell-tube wall) Main source of contamination during a sort are Doublets Differently labeled cells may be attached and be deflected in the same drop.

  13. Sample Preparation • Cells or particles in monodispersed suspension Suspension Cells: • Concentration: 1 x 106 – 1x107 • Low protein concentration Adherent Cells: • Lower concentration • Low protein concentration • All sample filtered • Collection tubes coated with FCS - Help prevent cell loss and improve cell viability

  14. CollectionDevices Sorting can be done into: • Tubes (15 ml, 5 ml or 1 ml) • Up to 4 populations at the same time • Plates (all types) • Slides • Petri dishes

  15. CellSortingApplications

  16. SortingImmunophenotipicpopulations CD3+ CD4+ CD25- CD25+ CD3+ CD4+ CD25- CD3+ CD3+ CD4+ CD3+ CD4+ CD25+ Transcriptomics (RNA) Genomics (DNA) Metabolomics (metabolites) Fluorescencemicroscopy FISH FunctionalStudies Etc.

  17. Interphase Anaphase EstablishingFluorescentCellLines Carina Santos (IMM) Cultured mCherrysignal Sorted Human hepatoma cell line Expressing α-tubulinfusedwithmCherry mCherrysignal mCherrysignal

  18. Chromosomesorting Human cell line with translocation between chromosome 2 and chromosome 17 Normal humancellline AT-rich DNA signal GC-rich DNA signal

  19. Establishment ofCell Clones Clone A Clone B Clone C Sort single cell into each well time

  20. WhatisFlowCytometry? Flow Cytometry Introduction to Flow Cytometry IGC Workshop uic • CellSorting(end) Telma Lopes IGC – Nov 12, 2009

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