Cell Sorting – The Basics

RMS Flow Cytometer Course 2005 Peter O’Toole [email protected]

Tel: 01904 328722

Flow Cytometry and Sorting

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What is sorting?

How do you sort? (mechanisms) Which mechanism(s) is/are best and how do they compare?

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Why sort, what are the applications, what can you do with sorted cells?

Cell Sorting: A Definition

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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?

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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.

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

ooo oo oo oo oo oo oo o o o o o o Laser beam 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.

o o o o o o ooooooo o o o o o Mechanical movement

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 (~10 6 /ml)

Electrostatic Sorting : Stream-in-air o o o o o o o o o o o o o o o o o o o o o o o o o + + +

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o o o+ o+

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oooo left o o o oo oo oooo waste right 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 red sort left, green or black sort right, 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).

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 Break off point How many drops in an equivalent length?

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.

Cell Sorting

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

Cell Sorting

• Fixed 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!

Electrostatic vs Mechanical

Processes up to 100,000 / second Sorts up to 300 / second (catcher) 1000/s wave Aerosols possible Fully contained 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).

No nozzle to block Only one ‘sort’ Usually requires a ‘concentrator’ Not applicable

Purity and Recovery Modes

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

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

• 0.5 Drop • 1 Drop • 1-2 Drop • 2 Drop • 3 Drop

Sort Envelope

1 Drop Envelope % Gated R8% R9% Pre-sort 68.3

27.7

Enrich mode Purify mode 7.4

91.9

0.33

99.5

Sort Results

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

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.

Electrostatic Sorting : Recovery and Purity

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

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?

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Most samples studied by flow cytometry

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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?

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.

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Aim for one cell in each droplet Charge three drops to ensure ‘red’ cell is sorted