The Cell Sorting Facility of the GSF-Institute of Molecular Immunoloy Joachim W. Ellwart

1. The Cell Sorting Facility of the GSF-Institute of Molecular Immunoloy Joachim W. Ellwart 21.06.2006 www.cell-sorter.de

2. Our service • We separate cells with the MoFlo. • We perform difficult flow cytometric measurements or test new flow cytometric methods. For that purpose we adapt the flow cytometer to the biological questions. • We combine flow cytometric and cell kinetic methods. • We develop new flow cytometric methods.

3. Principle of a drop in air sorter

4. Principle of FACS-sorting • Single cell suspension, e.g. blood cells or isolated tissue cells. • The properties of the target cells were stained by fluorescent dyes. • A piezoelectric crystal in the nozzle causes the stream with the cells to break into individual droplets. • The system is adjusted so that there is a low probability of more than one cell being in a droplet. • Just before the stream breaks into droplets the flow passes through the observation point where the fluorescence intensities of each cell are measured by the flow cytometer. At this point the cells for sorting were selected. • An electrical contact placed in the nozzle holder loads the abrupting stream at the moment of disruption. The charge is trapped on the droplet after it breaks from the stream. The charged droplets containing selected cells then move through an electrostatic field that diverts the droplets into containers based upon their charge. • The separated pure cell fractions are ready for futher measurements or experiments.

5. When should fluorescence activated cell sorting be used instead of bulk separation methods like panning, elutriation or magnetic bead separations? • When very high purity (95%-100%) of the target population is required. • For separations on the basis of internal staining e.g. of DNA, or of internal antigens, or fluorescent protein. • For enrichment of populations on the basis of surface receptor density. • For separation of populations that have a low density of receptors on their surface. • For separations on the basis of multicolor staining. • When single cell sorting is required. • When other separation methods fail. Bulk separation methods should be used when the starting cell number is greater than ~300 million cells.

6. It is possible to sort cells to retrieve subpopulations according to fluorescent markers e.g. • ofprotein expression, • DNA content, • cell function, • or other stainable properties of the cells. • And according to light scatter parameters, • or a combination of these parameters.

7. At our cell sorting facilitycells are sorted for • culture, creation of stable cell lines, • DNA analysis, PCR, • RNA analysis, FISH, micro array analysis, • morphological analysis by microscopy, • protein extraction, Western blot analysis, • single cell cloning, • for functional tests.

8. Examples of commonly used fluorescent dyes for cell sorting with our MoFlo • Immunophenotyping with 9-color sorting: e.g. Cascade Blue, Amca, FITC, CY3, PE, Cy5, APC, TR, Per-CP, Alexa-dyes, Tandem dyes. • Fluorescent protein expression such as: eBFP, eCFP, eGFP, eYFP, Ds-Red. • Cell division sorting by BUdR/Hoechst, CFSE or PKH26. • Cell-cycle and cell-ploidy sorting: PI, Hoechst dyes, DAPI. • Calcium mobilization sorting with Indo-1. • Apoptosis sorting of the sub diploid peak or according Annexin V-FITC staining. • Scatter parameters and autofluorescence. At our MoFlo almost all common fluorescent dyes can be used to mark subpopulations for sorting.

9. Typical Multi-Parameter Sorting: Immunophenotyping Characterisation of the regulatory function of CCR2+ T-cells in murine spleenocytes fluorescently labelled antibodies for the detection of specific antigens: CCR2-PE, CD4-APC, CD44-FITC Excitation 488 nm Fluorescence intensity 565 – 593 nm R4 Excitation 488 nm Fluorescence intensity 565 – 593 nm 0.2% CD44-FITC+ CCR2-PE+ CD4-APC + Excitation 647 nm Fluorescence intensity 660 – 680 nm Excitation 488 nm Fluorescence intensity 520 – 540 nm • Isolation of CD4+ CD44+ CCR2+ T-cells • Nessesary purity of >95% is achieved in one sorting step

10. Sort regions Boolean logic • inside a polygonal Region • outside • AND Look Up Table for each region • 256 x 256 channels

11. Sorting according fluorescent protein expression Ba/F3 cells, two muations in FLT3 gene linked with different fluorescent proteins Excitation 488 nm Fluorescence intensity 530 -550 nm 0.1% eGFP, eYFP and PI emission spectra and emission filters eYFP+ eGFP+ • FLT3-ITD-TKD dual mutant expressing cells were sorted • with high cell purity Fluorescence intensity 500 – 520 nm • for cell culture

12. Combination of DNA content and fluorescence protein expression (Hoechst 33342 and eGFP) Summit Software FlowJo Software G2M S G0G1 Excitation 364 nm Fluorescence intensity 430 – 470 nm Excitation 488 nm Fluorescence intensity 520 – 540 nm • Cells were sorted according their cell cycle distributionand transfection with a eGFP marked gene.

13. Stem cell sorting: side population (SP-cells) Excitation 364 nm fluorescence intensity 420 – 480 nm a G2M S G0G1 SP debris fluorescence intensity 655 – 685 nm g Murine bone marrow stained with Hoechst 33342 and propidium iodide Stem cells are known to have highly efficient pumps for Hoechst 33342. The emission wavelength shifts to the blue end of the spectrum when the dye concentration is low. SP-cell sorting can be combined with phenotyping.

14. high Indo-1 stainingExcitation 364 nm Sorting according the calcium content after cooling mast cells down from RT to 4°C Fl (390–430 nm) /Fl (515–545 nm) fluorescence intensity 390 – 430 nm low 0 min 10 min fluorescence intensity 515 - 545 nm temperature 24°C • The sorted cells were proved by PCR for the cold receptors TRPA1 (ANKTM1) and TRPM8 (CMR1). 4°C Time

15. General cell sorting parameters light scatter: 90-degree scatter intensity Gives information about the morphology of the cells. vitality: propidium iodide SSC fluorescence intensity 625 – 645 nm Forward scatter intensity doublet discrimination: forward scatter - area FSC - A As a cell dies it's plasma membrane becomes permeable allowing fluorescent dyes present outside the cell to enter it and fluoresce. pulse width Doublets will have greater pulse width than a single cell, as they take longer to pass through the laser beam, and therefore can be excluded from the sorting. forward scatter - area

16. Doublet discrimaination < 5.5µs two separate events t > 5.5µs coincidence abort Voltage t w doublet threshold t

17. Cell preparation for cell sorting with the MoFlo • prepare a single cell suspension • optimal concentration of about 20 - 40 million cells per ml • Ca2+/Mg2+ free media when working with sticky cells • to prevent clumps use not more than 0.5 % FCS or BSA • use cold buffers to prevent clumps • add 0.1mM EDTA to prevent clumps or use commercial cell dissociation media • avoid dead cells, debris, and other unnecessary events • use a Dead-cell-removal column (Pasteur pipette with cotton wool) • add 100µg/ml DNAse when working with broken cells • we use a Filter of 53 µm pore size to remove any clumps or aggregates • most fluorescent dyes are light sensitive – work in dimmed light

18. Technical description of our MoFlo • Typical sort rate is 10 - 30,000 starting cells per second. • 150.000 cells/s can be analysed by the electronics. • Number of sort populations is maximal four. • Single cell or multi well sorting can be performed. • Excitation laser wavelengths: from real UV (364nm) to deep red (676 nm) according our 2 Argon and one Kypton lasers. • Fluorochrome options: nearly all. We can accommodate the most available fluorophores. • Up to nine different colour analysis per cell can be performed. • Sort region type is polygonal. • The instrument is equipped with temperature controlled sample and collection chambers. • It is also equipped with an aerosol containment system for sorting of biohazardous samples (S2 and L2).   • We perform sterile separations.

19. Typical system parameters nozzle diameter 70 µm pressure 60 psi stream velocity 25 m/s droplets / s 100.000 Hz droplets / cell ~3 (at 30.000 cells/s) t / cell ~33 µs (at 30.000 cells/s) t / droplet 10 µs t / lymphocyte 0.4 µs dead time 5.5 µs

20. Collection tubes multiwell plates 6 – 384 wells Eppendorf tubes one to four tubes /populations 5 ml to 50 ml Falcon tubes onto slides or directly onto filters or nitrocellulose membranes. • The volume of one sorted drop is 1.4 nl. 106 cells result in 1.4 ml. • Give 1-2 ml serum into the collection tube or whatever your cells will be happiest in !

21. Sort modeaffects the number of aborts • Enrich Mode: all sorted drops with a positive cell are chosen regardless of contaminats. • Purify Mode: contaminating events in the sorted drops will result in an abort decision. • Single-Cell Mode: only drops containing one positive cell having a safe zone are sorted.

22. O O wu Sort modes O w O w enrich mode u purify mode °single cell mode O wu O O wu° O O w

23. Abort terminology • Hard aborts: 1) events that are not located in the logical sort windows. 2) when a second event is detected during the 5.5 µs of instrument dead time. • Software–defined aborts: events that do not match the defined sort mode.

24. Will the cells be harmed by the sorting process? • Generally, the cells will be not harmed through the process itself as long as they are maintained at a temperature, pH, and in media that is most suited to them. In most cases cells are at least 95% viable after a sort with typical system parameters. What comes out is closely related to what goes in the sorter.

25. Estimated sort times • Every sort session at the MoFlo takes about 90 min of set up time and 15 minutes of post-sort time. • The time of the cell separation process depends on the starting cell number and not on the target cell population. • Adherent cell lines: ~30 million unseparated cells/ h • Thymocytes/blood: ~100 million unseparated cells/ h • Sorting of 10x106 cells takes in all about 2h, of 100x106 about 3-5h. • A cell concentration lower than 30 x106/ml prolongs the actual sorting time. • The sorting time is enhanced needlessly by dead cells, debris and unwanted cells like erythrocytes.

26. What is the maximum purity of a population that can be achieved in single-cell mode and what does it depend on? • Maximum purity is 99% to 100%. Normally it is obtained in one sorting step when the target population is greater than 10%. • The sort purity generally exceeds 95% when the target population is greater than 1%. • The purity of sorted cell fraction depends on the quality of the sample (debris, free DNA, adherence). In general, the higher the purity of sorted cells is the lower is the yield. The purity is also dependent on the hydrodynamic flow cytometer stability.

27. Is it possible to sort a population that comprises less than 1% of the total or do I have to enrich it before the sort ? • Yes. Rare event sorts below 1% target population can be done but often have low purity and a low yield. Therefore, whenever possible, cells should be enriched through bulk methods or through a preceding enrichment sort.

28. How many cells do I need to prepare to yield 1 x 106 of a population that comprises 10% of the cells? • 1 x 106 = 10% target population x 50% yield x20.1 x106starting cell number - 0.1 x106 cells for adjustment. • 50% yield is a reasonable number, but the actual percentage of cells that are yield depends on a multitude of factors: • Cell death that occurs during, pre- and post sort • and loss through adherence of cells to tube walls (we use polypropylene instead of polystyrene tubes). • Sort rate: the higher the sort rate the lower the recovery. • Precision of sort set up and stability of the flow cytometer. • Sort mode: Enrichment sorts have higher yield than purity sorts. • The number of adjustment cells depends on the degree of difficulty of the flow cytometric measurement.

29. Important definitions number of target cells in the sorted sample total number of particles in thesorted sample number of target cells in the sorted sample number of target cells asindicated by the sorter number of target cells in the sorted sample number of target cells in the original sample Purity (%) = Recovery (%) = Yield (%) =

30. This makes the sort session more successful: • First inform us and discuss with us. The more we know the better for the experiment ! • If possible perform before sorting an analytical measurement at an analytical flow cytometer in order to check your staining. • Provide as many positive and negative controls as you can, one set for each color to adjust the positive and negative population. • Someone familiar with the samples needs to be reachable by phone during the sorting.

31. Discuss with the core staff • Inform us about the intent of the experiment, • Dye combinationg suitable optics, • Properties and preparation of the cells, cell number, necessary purity. g estimation of the sorting time. • What you will do with the cells after sorting? • Please contact us to schedule an appointment for cell sorting. • Pathogens (L2) or genetically manipulated material (S2) ? • Please complete the Cell Sorting Application Form which you will find in the internet: http://www.cellsorter.de. • The current sign up time for sorting is about 1 week. • Inform us as soon as possible about a Cancellation.

32. Access:The service is available by appointment. The MoFlo is operated by the MoFlo Specialists Karin Nispel and Joachim Ellwart. Users bring prepared samples and the flow cytometry specialists will sort them. To prevent mismanagement of the instrument by many different users the MoFlo needs to be handled only by us. The Cell Sorting Facility of the IMI-Institute

33. Distribution of users of the sorting facility in 2005 total: 144 sort sessions – 27 sort projects Bio-Companies 2% University Hospital Rechts der Isar 1% IMI 17% MPI of Neurobiology 25% Junior Research Groups 5% LMU Biozentrum 5% KMOLBI 23% University Hospitals LMU 4% GSF-Institutes Neuherberg 5% Dept. of Gene Expression 1% Clinical Cooperation Groups 12% GSF 63%

34. Last but not least: • Feedback about the outcome of your experiment, especially if things did not turn as expected, is essential for us. Please let us know and discuss alternative solutions with us.

35. Contact persons: Karin Nispel Joachim EllwartContact information: www.cell-sorter.de The Cell Sorting Facility of the Institute of Molecular Immunology at the Helmholtz Center for Environment and Health