1. Cell Sorting – The Basics RMS Flow Cytometer Course 2005
Peter O’Toole
Tel:

2. 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?

3. 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.

4. 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.

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

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

7. 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.

8. FACS piezo-electric sorter

9. 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)

10. 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 (+/ 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

12. Charged droplet sorter

13. Charged droplet sorter

14. 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?

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

16. 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

17. 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!

18. Phase gating

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

20. 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

21. 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

22. Drop Decision
All options will sort this cell

23. Drop Decision
Only enrich will sort this drop

24. Drop Decision
Enrich will sort this drop

25. Drop Decision
Purify and enrich will sort this drop

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

27. 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

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

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

30. 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.

31. 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

32. Sorting lymphocytes Before sorting After sorting Quad % Gated
(1)-UL
(2)-UR
(3)-LL
(4)-LR
Quad % Gated
(1)-UL 0.00
(2)-UR 0.00
(3)-LL 0.00
(4)-LR

33. 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

34. 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!!!

35. 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………………

36. 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

37. Any questions?

38. 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 +/ 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.

39. 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