Title: Cell Sorting The Basics RMS Flow Cytometer Course 2005
1Cell Sorting The BasicsRMS Flow Cytometer
Course 2005
- Peter OToole
- pot1_at_york.ac.uk
- Tel 01904 328722
2Flow 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?
3Cell 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.
4How 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.
5Mechanical Sorter
- FACS Calibur / FACS Sort
- Flow Activated Cell Sorting
Electrostatic Sorter
- MoFlo, Aria, Vantage, EPICS Elite
Pressure Differential Sorter
6Mechanical Sorting
- Piezo-electric deflector
- Fully enclosed (can sort hazardous sample)
- Relatively inexpensive
- Slow (300 sorts/sec)
7Mechanical Sorting Within a Flow Cell
ooooo ooooo ooooo ooo o Laser
beam o o o o o
o o o ooooooo
o o o o 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.
8FACS piezo-electric sorter
9Electrostatic Cell Sorting
- Charge drops non-mechanical
- More difficult to enclose (biohazard)
- Relatively expensive
- Fast (upto 100,000 sorts/sec)
- High end concentrations (106/ml)
10Electrostatic Sorting Stream-in-air
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.
Reproduced from Terry Hoy
11(No Transcript)
12Charged droplet sorter
13Charged droplet sorter
14Poor 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?
15Cell Sorting
- Droplet formation is governed by
- transducer frequency
- transducer amplitude
- nozzle diameter
- sheath pressure
- phase of the charging pulse
16Cell Sorting
- Fixed parameters
- transducer frequency (minor adjustment)
- nozzle diameter
- sheath pressure
- Adjustable parameters
- transducer amplitude
- phase of the charging pulse
- drop delay position
17Cell 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!
18Phase gating
19Cell Sorting
- Charging pulse must be in phase with transducer
Test pulse switched on Observe side streams
out of phase
In phase
20Electrostatic 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 No
t applicable
21Purity and Recovery Modes
Single Mode High Concentration
22Drop Decision
All options will sort this cell
23Drop Decision
Only enrich will sort this drop
24Drop Decision
Enrich will sort this drop
25Drop Decision
Purify and enrich will sort this drop
26Sort Envelope
- 0.5 Drop
- 1 Drop
- 1-2 Drop
- 2 Drop
- 3 Drop
27Sort Results
1 Drop Envelope
28Sort Results
29Cell Sorting
- Yield and purity
- compromise
- anti-coincidence off - high yield, lower purity
- anti-coincidence on - lower yield, high purity
30Electrostatic 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 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
32Sorting lymphocytes
After sorting
Before 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
33Sterile 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
34Sample 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!!!
35What 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
36What 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
37Any questions?
38Poor 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.
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