1. What is Flow Cytometry? Flow Cytometry uic April 05, 2013 Cell Sorting Flow Cytometry Workshop IGC

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

3. Cell Sorting (https://www.youtube.com/watch?v=W8mLBhQyJJI) Real Time Sorting Machine Made by: Petur Olsen, Thomas Bøgholm, and Henrik Kragh-Hansen Aalborg University, Denmark, 2007

4. Cell Sorters MoFlo (Beckman Coulter) Avalon (Propel Labs) FACSJazz (BD Biosciences) Influx (BD Biosciences) FACSAria (BD Biosciences) FACSVantage (BD Biosciences) JSAN (Bay Bioscience) SH800 (Sony) Synergy3200 (i-Cyt) Astrios (Beckman Coulter)

5. A cell sorter sorts drops, not cells! Charge Drop With particle of interest Drop Generation Break Stream into Droplets (30-100,000 drops/sec) Flow Sorting Flow Cell Drop Breakoff Nozzle cuvette Charge High Voltage Electric Plates Waste Collection Tube Deflect Charged Drop Physical separation of a cell or particle of interest from a heterogeneous suspension of cells or particles.

6. Drop Delay Flow Cell Drop Breakoff Nozzle cuvette Drop Delay Sorting relies on some statistical rules for randomly distributed events: Poisson Statistics These rules provide the theoretical background for calculating the achievable sort rates and sorting efficiency Drop Delay: Time between laser interception and drop breakoff

7. Limitations derived from Poisson Statistics # events/sec < 1/4 of # drops/sec 1 cell for every 4 drops (ensures that the probability of having more than 1 cell in a drop is very low) Example: At frequency of 90,000 drops/sec, rate should be lower than 22,500 events/sec Average: 1 cell/1 drop 1 cell/2 drops1 cell/4 drops1 cell/6 drops............ 1.1% 14% 2.4% 19.5% 7.6% 30% 18.4% 36.8%

8. Limitations derived from Poisson Statistics Cells slightly apart at the interrogation point may end up in the same drop. If sorting in purity mode, sorter will abort sort decision compromising recovery. Trailing drop Leading drop Interrogated drop Flow Cell Drop Breakoff Nozzle cuvette Drop Delay

9. If non-target particle is too close to the interrogated drop, the latter wont be sorted. Sort Precision: Purity Trailing dropLeading drop Interrogated drop Purity Mask

10. Sort Precision: Yield or Enrichment If a particle of interest is close to the edge an extra drop will be sorted. Trailing dropLeading drop Interrogated drop Yield Mask

11. Particles near the drop edge can affect the breakoff and alter the trajectory of the deflected drop. The sort decision will be aborted. Sort Precision: Single Cell Trailing dropLeading drop Interrogated drop Phase Mask

12. Cell sorting Performance Sorting Performance (Recovery, Purity, Speed) depends on: 1.Operator (how well machine is setup and calibrated) 2.Researcher/Clinician (sample preparation) Flow cytometers sort drops very accurately, it's your job to get the particle of interest into the sorted drop... Mark Dessing, BD Biosciences Anonymous operator monitoring sort experiments.

13. PropertyResearcher/ClinicianOperator Cell Dimension (diameter) Filter cells with appropriate mesh size. Nozzle size (DDF*, Pressure). Cell Type or tissue (morphology, fragility, stickiness) Sample Buffer Recipe. Optmize Concentration. Collection Buffer Recipe. Pressure (Nozzle size, DDF*). Staining (# colors, fluorochrome) Choose appropriate color panel. Use at least single-color compensation controls. Choose right laser/filter combination. Frequency of target populations Optimize concentration. Collection tubes can have larger volume of Media for better recovery. If rare events, may be best to enrich first and sort again in purity mode. Sorting type (Enrichment, Purity, Single-cell mode) Optimize concentration. Select appropriate sort decision mode. # Cells to sortDetermine total # cells to prepare.Book appropriate time for sorting. Collection (tube, plate, slide, etc) Coat collection receptacles. Use appropriate buffer volume. Prepare/calibrate appropriate sample collection. Communication between operator and researcher is ESSENTIAL If youre sorting, consider discussing the following issues with the operator: *DDF – Drop Drive Frequency (in Hz, indicates the number of drops generated per second

14. Sample Preparation

15. Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm 1.Single Cell Suspension. 2.Optimized Sample Concentration. 3.Appropriate Sort and Collection Buffer. 4.Expedient Sample Processing. Recovery Purity Speed Recovery Purity

16. Single Cell Suspension Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm 1.Avoid clump formation (see Sample Buffer Recipe) 2.Filter ALL samples just before sorting (mesh size 30-50μm). 3.Remove aggregates from sort gates with doublet discriminator. One of the most (if not the most) important aspects for attaining best Purity and Recovery is for cells to be in a single cell suspension before and during the sort. http://www.partec.com Unwanted cells may be attached and be deflected in the same drop compromising Purity. Doublet exclusion removes cells of interest, reducing Recovery.

17. Optimize Sample Concentration Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm 1.Coincidence Aborts. 2.Coefficient of Variation (CV) and Resolution. 3.Cell Adhesion and Clumping. There is no single ideal concentration for all cell types and sort setups. Optimal concentration must be found for best sorting performance.

18. Optimized Sample Concentration Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm 1.Coincidence Aborts. There is no single ideal concentration for all cell types and sort setups. Optimal concentration must be found for best sorting performance. If sample too concentrated, increased coincidence aborts will compromise Recovery Trailing drop Leading drop Purity Mask (FACSAria, BD) Interrogated drop

19. Optimized Sample Concentration Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm 2.Coefficient of Variation (CV) and Resolution. There is no single ideal concentration for all cell types and sort setups. Optimal concentration must be found for best sorting performance. If sample too diluted, Speed is compromised. If flow rate is increased to increase sort speed, resolution is compromised and consequently Recovery. Low Flow Rate Laser Sheath Sample High Flow Rate Laser Sheath Sample

20. Optimized Sample Concentration *Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm 3.Cell Adhesion and Clumping. There is no single ideal concentration for all cell types and sort setups. Optimal concentration must be found for best sorting performance. Sticky cells should be run slightly more diluted. Suggested Concentration Ranges* Nozzle SizeCell TypesConcentration (cells/mL) 70μmLymphocytes, thymocytes8-15 x 10 6 80μmActivated subsets, smaller cell lines7-10 x 10 6 100μmLarger adherent cells5-9 x 10 6

21. Sort Buffer Recipes *Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm 1x Phosphate Buffered Saline, PBS (Ca/Mg ++ free) 1mM EDTA 25mM HEPES pH 7.0 1-2% Fetal Bovine Serum, FBS (Heat Inactivated) Filter sterilize with 0.2µm mesh and store 4°C Again, one of the most important aspects to improve Purity and Recovery is a well designed buffer recipe to maintain cells in a single cell suspension. Basic Sorting Buffer*: Clean Lymphoid Cells Buffer can be simplified to Hanks Balanced Salt Solution (HBSS) with 1-2% FBS. No need for EDTA. Sticky Cells Raise concentration of EDTA to 5mM. Use FBS dialyzed against Ca/Mg ++ free PBS

22. Samples with high percentage of Dead Cells It is likely that there is soluble DNA from dead cells leading to severe clumping. Add 10U/mL DNAse II to the buffer recipe. Adherent Cells Use cation-free PBS/FBS buffer to inactivate trypsin. (cations promote adherence of cells to plate and to each other). Level of EDTA can be increased if necessary but too much EDTA can be deleterious. Sort Buffer Recipes *Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm 1x Phosphate Buffered Saline, PBS (Ca/Mg ++ free) 1mM EDTA 25mM HEPES pH 7.0 1-2% Fetal Bovine Serum, FBS (Heat Inactivated) Filter sterilize with 0.2µm mesh and store 4°C Again, one of the most important aspects to improve Purity and Recovery is a well designed buffer recipe to maintain cells in a single cell suspension. Basic Sorting Buffer*:

23. Collection Buffer Recipes *Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm Fetal Bovine Serum only. Your own culture media with at least 10% serum. PBS if collecting cells for RNA or DNA. Lysis buffer from RNA isolation kit Collection buffers are important to maintain cells in appropriate conditions during the sort and for enhanced viability and Recovery. Basic Collection Buffers*: For re-culturing cells, ALWAYS use antibiotics (pen-strep and gentamicin). One may also add antifungal agents. After cells are sorted into collection tubes, always centrifuge to remove diluted buffer and replenish with culture media.

24. Collection Buffer Recipes Collection buffers are important to maintain cells in appropriate conditions during the sort and for enhanced viability and Recovery. Sample Collection: Collection tubes/wells should have enough collection buffer to accomodate sorted cells. Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm 1ml Media in 5ml tubes. 3ml Media in 15ml tubes. 20-100μl in each well of 96-well plates. Tip: Leave tubes filled with Media (w/ 10% serum) 30min before starting sort. Remove Media leaving only the required amount for sort. This will coat the inner walls of the tube to prevent sorted cells from adhering and dehydrating before reaching the collection Media.

25. Expedient Sample Processing Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm The time to prepare cells for sorting should be as short as possible to minimize stress and maximize Recovery. 1.ALL reagents should be prepared BEFORE starting sample preparation. 2.ALL steps of the protocol should be optimized with minimal sample processing. 3.Usually, large amounts of material need to be prepared to obtain the appropriate number of collected cells. Dont be afraid to ask for help for sample preparation.

26. Representative Examples Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm Example 1 Researcher/Clinician needs 10 x 10 6 B220 + cells from a Lymph Node sample. Total # cells to sort: 10 x 10 6 ÷ 0.322 (32.2% B220 + cells) ÷ 0.80 (80% efficiency) = 39 x 10 6 total cells At a rate of ~20,000 events/sec, it will take slightly more than 30 minutes to sort this sample, assuming concentration has been optimized. Nozzle 70μm Drop Drive Frequency = 90,000 Hz. Purity Mode Sort into 15mL tube pre-coated with Media

27. Representative Examples Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm Example 2 Researcher/Clinician needs 10 x 10 6 Foxp3-GFP + cells from a Lymph Node sample. Total # cells to sort in enrich mode: 12 x 10 6 ÷ 1 (100% foxp3-GFP + cells) ÷ 0.99 (99% efficiency) = 12 x 10 6 total cells At a rate of ~20,000 events/sec, it will take around 10 minutes to sort this sample, assuming concentration has been optimized. Nozzle 70μm Drop Drive Frequency = 90,000 Hz. Trigger on GFP + cells Enrich Mode If Purity mode is used: 10 x 10 6 ÷ 0.025 (2.5% foxp3-GFP + cells) ÷ 0.75 (75% efficiency) = 535 x 10 6 total cells, i.e., at a rate of 20,000 events/sec it would take ~7.5 hours to sort this sample.

28. Representative Examples Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm Example 2 Researcher/Clinician needs 10 x 10 6 Foxp3-GFP + cells from a Lymph Node sample. Total # cells to sort in purity mode: 10 x 10 6 ÷ 0.1 (10% foxp3-GFP + cells) ÷ 0.75 (75% efficiency) = 135 x 10 6 total cells At a rate of ~20,000 events/sec, it will take slightly less than 2 hours to sort this sample, assuming concentration has been optimized. Adding an extra 20 minutes to centrifuge enriched population, it would take approximately 2h10min to sort this sample (~4-fold less than using Purity mode from the beginning). Nozzle 70μm Drop Drive Frequency = 90,000 Hz. Purity Mode Sort into 5mL tube pre-coated with Media.

29. Sample Preparation (summary) Based on Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm Sample preparation is by far the most important factor influencing sorting performance. 1.Anything affecting single cell suspension will compromise Purity.. To safeguard purity, then Recovery is compromised. 2.Cell size, morphology, fragility,and concentration may affect Speed.. 3.Everything can compromise Recovery. Recovery Purity Speed Recovery

30. Cell Sorting Applications

31. Transcriptomics (RNA) Genomics (DNA) Metabolomics (metabolites) CD3+CD3+ CD4+ CD3+ CD4+ CD25- CD3+ CD4+ CD25+ CD3+ CD4+CD25-CD25+ Fluorescence microscopy FISH Functional Studies Etc. Sorting Immunophenotipic populations

32. Interphase Anaphase Human hepatoma cell line Expressing α-tubulin fused with mCherry mCherry signal Sorted Cultured Carina Santos (IMM) Establishing Fluorescent Cell Lines

33. Chromosome sorting Human cell line with translocation between chromosome 2 and chromosome 17 Normal human cell line GC-rich DNA signal AT-rich DNA signal

34. Establishing Cell Clones Sort single cell into each well time Clone A Clone B Clone C

35. What is Flow Cytometry? Flow Cytometry uic Cell Sorting, end Sources: Guidelines for Cell Sorting: http://www.cincinnatichildrens.org/research/cores/flow-cyto/cellsorting.htm Optimizing Sorting Experiments: http://facs.scripps.edu/SortLink/sortprep.html Flow Cytometry Workshop IGC