WIXLIB

Cell SortingAn overviewMarcus Eich

Possible applications RNA isolation, Single cell qPCR Protein isolation Discrimination of transfected cells (e.g. GFP) Single cell cloning Synchronization (Stem) Cell transplantation 4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Cytometer basicsWasteprime (PBS)Valvelaminar flowLaserInterrogation pointObjective of optical Bench(Condenser)Sheath (PBS)Air-pressureCollection -lenseLaserblockerscattered lightQuartz-cuvettePhotodiodeValveVacuum (prime)Sample-Injection needle / port (SIP)Tubeby Klaus Hexel4/21/2016 Marcus Eich, Steffen SchmittSingle-cell suspensionHI-STEMCore Facility Flow Cytometry

Principle of cell sorters“Cuvette”“Jet in thstained cellsstained cellshydrodynamic focushydrodynamic focus„nozzle“fluorescence signalsfluorescence signalslaserbeamlaserbeamlight scattercuvette„nozzle“light scatterdroplet break offdroplet break off- - deflected dropsdeflected dropssorted cellssorted cellswaste4/21/2016 Marcus Eich, Steffen SchmitttransducerwasteHI-STEMCore Facility Flow Cytometry

Principle of cell ained cellshydrodynamic focusfluorescence signalslaserbeamlight scattercuvette„nozzle“droplet break off- deflected dropssorted cellswaste4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Principle of cell ained cellshydrodynamic focusfluorescence signalslaserbeamlight scatterKüvette„nozzle“droplet break off- deflected dropssorted cellswaste4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

The nozzles 70 µm 87 KHz (87.000 drops/s)Sortspeedincreases 85 µm 47.7 KHz (47.700 drops/s)Cellviabilityincreases 100 µm 28.2 KHz (28.200 drops/s)Nozzle diameter/3 biggest cell size for sortingNozzle usage depends on downstream assay4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Stream & Drop DelayHow to set up the streamGap Set stream to a stable drop1 positionhaving a constant gap by modulation ofthe phase and amplitude.Drop1 Define drop delay under these constantconditions.Drop DelayDistance from interrogationpoint to drop1 position4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Droplet characteristicsCalculation by Tobias Rubner4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Distributions of cells in streamIn reality there is no equaldistribution of particles.A random distribution isreality.Under this condition evenrare particles could e, it becomesdifficult to find rare eventscompletelyfreeofunwanted companions.4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Max. sort speed with 70 µM25.000 cells / sec1.5 x 106 cells / min9 x 107 cells / h2,1 x 109 cells / dThat means for a 0.1% population 2x 106 cells / d4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Possible targets 4 populations 4 populations 2 populations 5 ml FACS tubes 1.5 ml tubes 15 ml tubes 6-well plates 24-well plates 96- well plates 384-well plates Glas slides Continuous cells Specific cell number4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

LimitationsYieldPurityTimeYieldPurityTimeTime Purity Yield YieldTimeTimePurity Flowrate4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

What is the aim of the sort?Before cell sorting some considerations souldto be made: Which downstream assay is planned? What is the desired number of cells? Is high purity an issue? How may cells do you have to start with? What is the percentage of cells of interest? Is pre-enrichment or depletion (MACS, Ficoll,etc.) possible?4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Calculation of the yield10% cells of interest are in the sort gate100.000 cells are analysed10.000 cells could have been sorted9.000 cells were sorted into the tubeYield is 90%4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

RecoveryMostly:70% - 80% of the sort countsReason: Cells die and get fragmented Cell sorting is a very stressfulprocess for the cell Cells stick to the tube Material of target is important Sample processing after sortingWhy does the cell sorter not detect all cells, which were countedafter harvesting? Loss of cells during sample preparation before sorting:Centrifugation, Transfer, Washing, Errors during counting4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Predefined Sort optionsSort precision mode: Yield low purity depending on theflow rate Purity low yield depending on theflow rate Single Cell highest purity, very low yield Determined by sort masks4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Sort-Masks There are 3 types of sort masks, which in combination define thesort precision modes:1 Yield mask Purity mask Phase mask32 Every drop is virtually divided in 32 segments. A cell can be located in one or more ogf these segments The relative position of the cell inside the drop influences the sortdecision.4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Yield MaskSort envelopeDeterminination of number of sorted drops, Non-target cells are not consideredOnly the calculateddrop is sorted016sortIf the cell lies atThe border of thedrop, 2 drops aresortedsortsortsort8sortsortsort8sortAlways 2 dropsare sorted324/21/2016 Marcus Eich, Steffen Schmitt1616sortsort16sort16HI-STEMsortCore Facility Flow Cytometry

Purity MaskSort envelope016Control for particles in neighboring drop, Non-target cells are consideredNo coincidence:Cell is sortedregardless thepresence of anunwanted cellCells in theneighboring dropcause an abort(coincidence)sortsort8no sortno sortsortsort832Cells in theneighboring dropcause an abort(coincidence)16no sortno sort164/21/2016 Marcus Eich, Steffen SchmittHI-STEMNon-Target cellslocated in thesame drop like(sort envelope16/32)Core Facility Flow Cytometry

Phase MaskSort envelope01632Cell position inside a drop determines the sort decision, Non-target cells are not consideredPosition of a cellinside a drophas noinfluence ofsort decisionIf cell is in themiddle ofthe drop,drop issortsortedNo cell is sorted,because it isalways in thephase masksortsortsort881616no sortno sortno sortno sortno sort4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

SortprecisionYieldPuritySingle cellExplanationYieldmaskAlways two drops aresorted regardless ofcontaminating cells. Nocells are lost.32If possible two dropsaresortedwithoutcontamination of nontarget cells in theneighboring drop.32Drop is sorted, if the cellis in the middle of adrop. If the cell lies inthe border a sort abortoccurs.4/21/2016 Marcus Eich, Steffen rtsortsort32sort0sort032HI-STEM16sortCore Facility Flow Cytometry

How to increase the sort quality Duplet exclusion Influence of target medium during reanalysis Multiple thresholds Reanalysis of „negative“ cells4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

1. Gating out DupletsSort P5 P2 (Scatter Fluorescence)1% Target population89% Purity(not good enough!)4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

1. Gating out DupletsP5 P6 P7 P2 (Scatter Duplets Fluorescence)1% Target population99% Purity4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

2. Influence of target medium4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Re-Analysis of sorted events4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Control: FCS alone4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Reanalysis without FCS4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

3. Multiple ThresholdsThresholds can be set for each parameter separately.These can be combined multiple thresholds.5%0.1%SSC45%FSCFITCPE-Cy5by Jochen Barths4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

3. Multiple ThresholdsThresholds can be set for each parameter separately.These can be combined multiple thresholds.90%45%0.1%0.2%SSC10%5%FSCFITCPE-Cy5by Jochen Barths4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

3. Multiple ThresholdsThresholds can be set for each parameter separately.These can be combined multiple y5by Jochen Barths4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

3. Multiple ThresholdsThresholds can be set for each parameter separately.These can be combined multiple E-Cy5by Jochen Barths4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

3. Multiple ThresholdsAdvantages Analysis only of the cells of interest at a faster rate Rare cell populations become better visible on the plots High throughput sorting Isolation of rare cells at high speed with high yieldDisadvantage Purity cannot be achieved, because events under thethresholds are not analysed and evaluated for the sortdecision4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

4. Another sort example . . .4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Reanalysis: Is it pure ?SignalBackground4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Index sort Sorting of single cells into single wells. Detection of the sorted cells on histograms, plots and inthe statistics4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Index sort Sorting of single cells into single wells. Detection of the sorted cells on histograms, plots and inthe statistics4/21/2016 Marcus Eich, Steffen SchmittHI-STEMCore Facility Flow Cytometry

Before cell sorting some considerations sould to be made: Which downstream assay is planned? What is the desired number of cells? Is high purity an issue? How may cells do you have to start with? What is the percentage of cells of interest? Is p