Cell Sorting – The Basics RMS Flow Cytometer Course 2005 Peter O’Toole pot1@york.ac.uk Tel: 01904 328722

Flow Cytometry and Sorting What is sorting? How do you sort? (mechanisms) Which mechanism(s) is/are best and how do they compare? Why sort, what are the applications, what can you do with sorted cells?

Cell Sorting: A Definition The ability to select any population defined by a logical combination of regions (a “gate”) and isolate this population from the sample.

How Do You Sort Cells? Preliminary processes are the same as for analysis : 1) Hydrodynamic focusing of a mixture of cells or particles to form a central core within a fluid sheath. 2) Interrogation of the cells by a laser source with subsequent analysis of scatter and fluorescence signals. 3) Application of regions and gates to define sub-populations.

Pressure Differential Sorter Mechanical Sorter FACS Calibur / FACS Sort Flow Activated Cell Sorting Electrostatic Sorter MoFlo, Aria, Vantage, EPICS Elite Pressure Differential Sorter Galaxy

Mechanical Sorting Piezo-electric deflector Fully enclosed (can sort hazardous sample) Relatively inexpensive Slow (300 sorts/sec)

Mechanical Sorting Within a Flow Cell ooooo ooo o Laser beam o o o o o o o ooooooo o Mechanical movement Hydrodynamic focusing and interrogation takes place in a flow cell, when a sort decision is made a ‘catcher’ tube moves into the stream to collect the cell.

FACS piezo-electric sorter

Electrostatic Cell Sorting Charge drops – non-mechanical More difficult to enclose (biohazard) Relatively expensive Fast (upto 100,000 sorts/sec) High end concentrations (~106/ml)

Reproduced from Terry Hoy Electrostatic Sorting : Stream-in-air o o o o o o o o o o o o o + - Hydrodynamic focusing in a nozzle vibrated by a transducer produces a stream breaking into droplets. Laser interrogation and signal processing followed by sort decision : white sort right, red sort left, green or black no sort. Electronic delay until cell reaches break off point. Then the stream is charged : + if white - if red. - + Charged droplets deflect by electrostatic field from plates held at high voltage (+/- 3000 volts). o- o+ o oooo oooo oooo left waste right Various collection devices can be attached : tubes, slides, multi-well plates. Reproduced from Terry Hoy

Charged droplet sorter

Charged droplet sorter

Poor Recovery and Purity: Calibrating the drop delay Measurement Measure this distance L As the frequency is known the time taken for a cell to traverse L can be calculated. This time determines when to charge the stream as the required cell breaks off in a drop. Break off point How many drops in an equivalent length?

Cell Sorting Droplet formation is governed by transducer frequency transducer amplitude nozzle diameter sheath pressure phase of the charging pulse

Cell Sorting Fixed parameters Adjustable parameters transducer frequency (minor adjustment) nozzle diameter sheath pressure Adjustable parameters transducer amplitude phase of the charging pulse drop delay position

Cell Sorting Problems with droplet formation temperature viscosity of sheath fluid is temperature dependent install air conditioning dirt in/on the nozzle complete or partial blockage of the flow cell A clean sorter is a happy sorter!

Phase gating

Cell Sorting Charging pulse must be in phase with transducer Test pulse switched on Observe side streams In phase out of phase

Electrostatic vs Mechanical Processes up to 100,000 / second Aerosols possible Nozzle prone to blocking At least two ‘sorts’ (left / right) Cells available at a reasonable density Can sort into tubes, onto slides or into multiple well plates. Cloning (single cells). Sorts up to 300 / second (catcher) 1000/s wave Fully contained No nozzle to block Only one ‘sort’ Usually requires a ‘concentrator’ Not applicable

Purity and Recovery Modes Aim Result Enrich Get all positives High Recovery Purify Positives only High Purity Mixed Mode Sorts pure to right, aborts to left High purity and recover aborts Single Mode – High Concentration

Drop Decision All options will sort this cell

Drop Decision Only enrich will sort this drop

Drop Decision Enrich will sort this drop

Drop Decision Purify and enrich will sort this drop

Sort Envelope 0.5 Drop 1 Drop 1-2 Drop 2 Drop 3 Drop

Sort Results 1 Drop Envelope % Gated R8% R9% Pre-sort 68.3 27.7 Enrich mode 7.4 91.9 Purify mode 0.33 99.5

Sort Results % Gated R8 R9 Pre-sort Enrich mode Purify mode

Cell Sorting Yield and purity compromise anti-coincidence off - high yield, lower purity anti-coincidence on - lower yield, high purity

Electrostatic Sorting : Stream-in-air Three drops are normally charged for each sort decision to ensure cell is in a charged droplet + This situation causes the sort to be aborted if purity is required For high purity the number of vacant drops is increased before the ‘no abort’ decision is taken 99% is possible For high yield and moderate purity the number of vacant droplets can be lowered before ‘no abort’ 80% is possible For enrichment the abort mechanism can be turned off. Aiming for one cell per drop and sorting three droplets per sort decision will give an enrichment to 33% however low the starting frequency.

Low purity, Good recovery Electrostatic Sorting : Recovery and Purity + + + + + + ON Abort OFF Good purity and recovery Both improve with more vacant drops BUT sorting speed drops Low purity, Good recovery ‘blue’ cell may be in top +ve drop Good purity Low recovery

Sorting lymphocytes Before sorting After sorting Quad % Gated (1)-UL 24.91 (2)-UR 0.83 (3)-LL 26.34 (4)-LR 47.91 Quad % Gated (1)-UL 0.00 (2)-UR 0.00 (3)-LL 0.00 (4)-LR 100.00

Sterile sorting Run 70% ethanol or dilute bleach through sheath lines Use sterilised sheath fluid Swab areas around collection area with 70% ethanol Run 70% ethanol, then sterilised sheath fluid through sample line

Sample Requirements Healthy Cells – check by standard flow Precoated tubes Sterile Cells? Temperature for cells (37 or 4 C) Time out of incubator Concentration of cells Total number of cells Cell aggregation Stained Cells!!!

What can be sorted? Most samples studied by flow cytometry Cellular parameters measurable by flow cytometry. E.g. Intrinsic differences; size,shape,cytoplasmic granularity, autofluorescence and pigmentation. Extrinsic : DNA content / composition, RNA, protein, sulphydryl groups, antigens, cytoskeletal components, membrane structure (potential, permeability & fluidity), enzyme activity, endocytosis, surface charge,receptors, bound and free calcium, apoptosis, necrosis, pH, drug kinetics………………

What can you do with the cells? Culture Bone marrow and peripheral blood Single cells (cloning). Cells transfected with marker genes. Chromosomes. Cell depletion (negative sorting) DNA studies – PCR Protein studies – Mass spectroscopy Imaging – Cellular structure and fine detail

Any questions?

Poor Recovery and Purity: Calibrating the drop delay The drop delay may need recalibrating if recoveries and purities are lower than expected. This can be achieved by collecting a few events at the measured drop delay and at intervals on either side. Most machines work in quarter drop intervals. By measuring the recovery at each position any error between the measured delay and optimum delay can be determined. Typically a plateau will be present approximately +/- 0.5 drops either side of the optimum position.Beyond this region the recovery will fall rapidly. A few fluorescent beads can be sorted onto a slide and examined under a microscope or 1000 or so cells collected into a tube and reprocessed on the sorter. Some machines are very stable and rarely require this check. On others it may have to be carried out prior to each sort.

High Speed Enrichment of Minor Populations Sorter is triggered only by ‘red’ cells. ‘Blue’ cells are below the threshold and ignored by the electronics. Enrichment to 33% is possible however small the starting population The effective rate will be at the drop drive frequency i.e.20-50,000/sec. Aim for one cell in each droplet + + Charge three drops to ensure ‘red’ cell is sorted