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What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Flow Cytometry uic Cell Sorting IGC – Nov 12, 2009 Outline • Cell Sorters • • • • • Drop Formation, Charging, and Deflection. Drop Delay. Sort-Drop Decisions Purity and Recovery Sample preparation • Applications Cell Sorters FACSAria • • • • • MoFlo • 100,000 events per second • Blue, Red, UV, Yellow, Green lasers (only 3 at the time) • 8 simultaneous colors • Stream in air • Lasers aligned every time machine is switched on 100,000 events per second Blue, Red, Violet lasers 9 simultaneous colors Cuvette flow cell Fixed Alignment 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! Drop Formation, Charging, and Deflection • First you need to know what you want to sort Flowcell Charge Collection Tubes Aspirator Tube If cell of interest is within the gate at time of laser interrogation sorter waits until cell reaches the last drop (breakoff point) and charges the drop. Sheath fluid must be a conductive ionic solution Drop Delay At the moment of cells analysis (interrogation point) the cell is not in a drop All decisions are made on a theoretical drop at the interrogation point. Flow Cell cuvette Nozzle Stream is subject to a vibration with a certain frequency Drop Delay Formation of drops stabilized Drop Breakoff Distan between the interrogation point and the breakoff point constant From the drop delay information the system can calculate the position where the cell will be at the moment of droplet breakoff 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 Reality Flow Cell Sorting relies on some statistical rules for randomly distributed events: cuvette Nozzle Poisson Statistics Drop Delay Drop Breakoff These rules provide the theoretical background for calculating the achievable sort rates and the effectiveness of sorting Reality 1. Number of events/sec cannot exceed 1/4 of number of drops/sec Flow Cell cuvette Nozzle 1 cell for every 4 drops (ensures that the probability of having more than 1 cell in a drop is almost zero) 2. Cells slightly appart at the interrogation point may end up in the same drop. If sorting in purity mode, sorter will abort sort decision compromising recovery. Drop Delay Drop Breakoff Sort-Drop Decisions A sort decision must be made for each particle. The sort decision is based on the sort gates AND sort modes If sorting in Purity mode: • Decisions will compromise recovery If sorting in Recovery mode: • Decisions will compromise purity If sorting in Single-Cell mode: • High count accuracy Purity and Recovery 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. 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 Collection Devices 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 Cell Sorting Applications Sorting Immunophenotipic populations CD3+ CD4+ CD25- CD25+ CD3+ CD4+ CD25CD3+ Transcriptomics (RNA) Genomics (DNA) Metabolomics (metabolites) CD3+ CD4+ CD3+ CD4+ CD25+ Fluorescence microscopy FISH Functional Studies Etc. Establishing Fluorescent Cell Lines Carina Santos (IMM) Interphase Cultured mCherry signal Anaphase Human hepatoma cell line Expressing α-tubulin fused with mCherry mCherry signal mCherry signal Chromosome sorting Human cell line with translocation between chromosome 2 and chromosome 17 AT-rich DNA signal Normal human cell line GC-rich DNA signal Establishment of Cell Clones Clone A Clone B Clone C Sort single cell into each well time What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Flow Cytometry uic Cell Sorting (end) IGC – Nov 12, 2009