P1252109105QDyvI

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The Cell Sorting Facility of the GSF-Institute of
Molecular Immunoloy Joachim W. Ellwart
21.06.2006 www.cell-sorter.de
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Our service

• We separate cells with the MoFlo.
• We perform difficult flow cytometric measurements
  or test new flow cytometric methods. For that
  purpose we adapt the flow cytometer to the
  biological questions.
• We combine flow cytometric and cell kinetic
  methods.
• We develop new flow cytometric methods.

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Principle of a drop in air sorter
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• Principle of FACS-sorting
• Single cell suspension, e.g. blood cells or
  isolated tissue cells.
• The properties of the target cells were
  stained by fluorescent dyes.
• A piezoelectric crystal in the nozzle causes
  the stream with the cells to break into
  individual droplets.
• The system is adjusted so that there is a low
  probability of more than one cell being in a
  droplet.
• Just before the stream breaks into droplets
  the flow passes through the observation point
  where the fluorescence intensities of each cell
  are measured by the flow cytometer. At this point
  the cells for sorting were selected.
• An electrical contact placed in the nozzle
  holder loads the abrupting stream at the moment
  of disruption. The charge is trapped on the
  droplet after it breaks from the stream. The
  charged droplets containing selected cells then
  move through an electrostatic field that diverts
  the droplets into containers based upon their
  charge.
• The separated pure cell fractions are ready
  for futher measurements or experiments.

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• When should fluorescence activated cell sorting
  be used instead of bulk separation methods like
  panning, elutriation or magnetic bead
  separations?
• When very high purity (95-100) of the target
  population is required.
• For separations on the basis of internal staining
  e.g. of DNA, or of internal antigens, or
  fluorescent protein.
• For enrichment of populations on the basis of
  surface receptor density.
• For separation of populations that have a low
  density of receptors on their surface.
• For separations on the basis of multicolor
  staining.
• When single cell sorting is required.
• When other separation methods fail.

Bulk separation methods should be used when the
starting cell number is greater than 300
million cells.
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• It is possible to sort cells to retrieve
  subpopulations according to fluorescent markers
  e.g.
• of protein expression,
• DNA content,
• cell function,
• or other stainable properties of the cells.
• And according to light scatter parameters,
• or a combination of these parameters.

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At our cell sorting facilitycells are sorted for

• culture, creation of stable cell lines,
• DNA analysis, PCR,
• RNA analysis, FISH, micro array analysis,
• morphological analysis by microscopy,
• protein extraction, Western blot analysis,
• single cell cloning,
• for functional tests.

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Examples of commonly used fluorescent dyes for
cell sorting with our MoFlo

• Immunophenotyping with 9-color sorting e.g.
  Cascade Blue, Amca, FITC, CY3, PE, Cy5, APC, TR,
  Per-CP, Alexa-dyes, Tandem dyes.
• Fluorescent protein expression such as eBFP,
  eCFP, eGFP, eYFP, Ds-Red.
• Cell division sorting by BUdR/Hoechst, CFSE or
  PKH26.
• Cell-cycle and cell-ploidy sorting PI, Hoechst
  dyes, DAPI.
• Calcium mobilization sorting with Indo-1.
• Apoptosis sorting of the sub diploid peak or
  according Annexin V-FITC staining.
• Scatter parameters and autofluorescence.

At our MoFlo almost all common fluorescent dyes
can be used to mark subpopulations for sorting.
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Typical Multi-Parameter Sorting Immunophenotyping
Characterisation of the regulatory function of
CCR2 T-cells in murine spleenocytes
fluorescently labelled antibodies for the
detection of specific antigens CCR2-PE, CD4-APC,
CD44-FITC
Excitation 488 nm Fluorescence intensity 565
593 nm
R4
Excitation 488 nm Fluorescence intensity 565
593 nm
0.2
CD44-FITC CCR2-PE
CD4-APC
Excitation 647 nm Fluorescence intensity 660
680 nm
Excitation 488 nm Fluorescence intensity 520
540 nm

• Isolation of CD4 CD44 CCR2 T-cells

• Nessesary purity of gt95 is achieved in one
  sorting step

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Sort regions

• Boolean logic
• inside a polygonal Region
• outside
• AND
• Look Up Table for each region
• 256 x 256 channels

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Sorting according fluorescent protein expression
Ba/F3 cells, two muations in FLT3 gene linked
with different fluorescent proteins
Excitation 488 nm
Fluorescence intensity 530 -550 nm
0.1
eYFP
eGFP, eYFP and PI emission spectra and emission
filters
eGFP
FLT3-ITD-TKD dual mutant expressing cells were
sorted

• with high cell purity

Fluorescence intensity 500 520 nm

• for cell culture

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(Hoechst 33342 and eGFP)
Combination of DNA content and fluorescence
protein expression
Summit Software
FlowJo Software
G2M S G0G1
Excitation 364 nm Fluorescence intensity 430
470 nm
Excitation 488 nm Fluorescence intensity 520
540 nm

• Cells were sorted according their cell cycle
  distribution and transfection with a eGFP marked
  gene.

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Stem cell sorting side population (SP-cells)
Excitation 364 nm
fluorescence intensity 420 480 nm a
G2M
S
G0G1
SP
debris
fluorescence intensity 655 685 nm g
Murine bone marrow stained with Hoechst 33342 and
propidium iodide
Stem cells are known to have highly efficient
pumps for Hoechst 33342. The emission wavelength
shifts to the blue end of the spectrum when the
dye concentration is low. SP-cell sorting can be
combined with phenotyping.
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Sorting according the calcium content after
cooling mast cells down from RT to 4C
high
Indo-1 staining Excitation 364 nm
Fl (390430 nm) /Fl (515545 nm)
fluorescence intensity 390 430 nm
low
10 min
0 min
fluorescence intensity 515 - 545 nm
temperature
24C

• The sorted cells were proved by PCR for the
  cold receptors TRPA1 (ANKTM1) and TRPM8 (CMR1).

4C
Time
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General cell sorting parameters
light scatter
90-degree scatter intensity
Gives information about the morphology of the
cells.
vitality propidium iodide
SSC
fluorescence intensity 625 645 nm
doublet discrimination
Forward scatter intensity
forward scatter - area
FSC - A
As a cell dies it's plasma membrane becomes
permeable allowing fluorescent dyes present
outside the cell to enter it and fluoresce.
pulse width
Doublets will have greater pulse width than a
single cell, as they take longer to pass through
the laser beam, and therefore can be excluded
from the sorting.
forward scatter - area
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Doublet discrimaination
lt 5.5µs
two separate events
t
gt 5.5µs
coincidence abort
Voltage
t
w
doublet
threshold
t
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Cell preparation for cell sorting with the MoFlo

• prepare a single cell suspension
• optimal concentration of about 20 - 40 million
  cells per ml
• Ca2/Mg2 free media when working with sticky
  cells
• to prevent clumps use not more than 0.5 FCS or
  BSA
• use cold buffers to prevent clumps
• add 0.1mM EDTA to prevent clumps or use
  commercial cell dissociation media
• avoid dead cells, debris, and other unnecessary
  events
• use a Dead-cell-removal column (Pasteur pipette
  with cotton wool)
• add 100µg/ml DNAse when working with broken
  cells
• we use a Filter of 53 µm pore size to remove any
  clumps or aggregates

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Technical description of our MoFlo

• Typical sort rate is 10 - 30,000 starting cells
  per second.
• 150.000 cells/s can be analysed by the
  electronics.
• Number of sort populations is maximal four.
• Single cell or multi well sorting can be
  performed.
• Excitation laser wavelengths from real UV
  (364nm) to deep red (676 nm) according our 2
  Argon and one Kypton lasers.
• Fluorochrome options nearly all. We can
  accommodate the most available fluorophores.
• Up to nine different colour analysis per cell can
  be performed.
• Sort region type is polygonal.
• The instrument is equipped with temperature
  controlled sample and collection chambers.
• It is also equipped with an aerosol containment
  system for sorting of biohazardous samples (S2
  and L2).  
• We perform sterile separations.

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Typical system parameters
nozzle diameter 70 µm pressure 60
psi stream velocity 25 m/s droplets / s
100.000 Hz droplets / cell 3 (at 30.000
cells/s) t / cell 33 µs (at 30.000
cells/s) t / droplet 10 µs t /
lymphocyte 0.4 µs dead time 5.5 µs
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Collection tubes
multiwell plates 6 384 wells
Eppendorf tubes
one to four tubes /populations
5 ml to 50 ml Falcon tubes
onto slides or directly onto filters or
nitrocellulose membranes.

• The volume of one sorted drop is 1.4 nl. 106
  cells result in 1.4 ml.
• Give 1-2 ml serum into the collection tube or
  whatever your cells will be happiest in !

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Sort modeaffects the number of aborts

• Enrich Mode all sorted drops with a positive
  cell are chosen regardless of contaminats.
• Purify Mode contaminating events in the sorted
  drops will result in an abort decision.
• Single-Cell Mode only drops containing one
  positive cell having a safe zone are sorted.

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O
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wu
Sort modes
O
w
O
w enrich mode u purify mode single cell
mode
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wu
O
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wu
O
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w
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Abort terminology

• Hard aborts
• 1) events that are not located in the
  logical sort windows.
• 2) when a second event is detected during the
  5.5 µs of instrument dead time.
• Softwaredefined aborts
• events that do not match the defined sort mode.

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Will the cells be harmed by the sorting process?

• Generally, the cells will be not harmed through
  the process itself as long as they are maintained
  at a temperature, pH, and in media that is most
  suited to them. In most cases cells are at least
  95 viable after a sort with typical system
  parameters.
• What comes out is closely related to what
  goes in the sorter.

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Estimated sort times

• Every sort session at the MoFlo takes about 90
  min of set up time and 15 minutes of post-sort
  time.
• The time of the cell separation process
  depends on the starting cell number and not on
  the target cell population.
• Adherent cell lines 30 million unseparated
  cells/ h
• Thymocytes/blood 100 million unseparated
  cells/ h
• Sorting of 10x106 cells takes in all about 2h,
  of 100x106 about 3-5h.
• A cell concentration lower than 30 x106/ml
  prolongs the actual sorting time.
• The sorting time is enhanced needlessly by dead
  cells, debris and unwanted cells like
  erythrocytes.

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What is the maximum purity of a population that
can be achieved in single-cell mode and what does
it depend on?

• Maximum purity is 99 to 100.
  Normally it is obtained in one
  sorting step when the target population is
  greater than 10.
• The sort purity generally exceeds 95 when the
  target population is greater than 1.
• The purity of sorted cell fraction depends on the
  quality of the sample (debris, free DNA,
  adherence). In general, the higher the purity of
  sorted cells is the lower is the yield. The
  purity is also dependent on the hydrodynamic flow
  cytometer stability.

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Is it possible to sort a population that
comprises less than 1 of the total or do I have
to enrich it before the sort ?

• Yes. Rare event sorts below 1 target population
  can be done but often have low purity and a low
  yield. Therefore, whenever possible, cells should
  be enriched through bulk methods or through a
  preceding enrichment sort.

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How many cells do I need to prepare to yield 1 x
106 of a population that comprises 10 of the
cells?

• 1 x 106 10 target population x 50 yield x
  20.1 x106 starting cell number - 0.1 x106 cells
  for adjustment.
• 50 yield is a reasonable number, but the actual
  percentage of cells that are yield depends on a
  multitude of factors
• Cell death that occurs during, pre- and post sort
  
• and loss through adherence of cells to tube walls
  (we use polypropylene instead of polystyrene
  tubes).
• Sort rate the higher the sort rate the lower the
  recovery.
• Precision of sort set up and stability of the
  flow cytometer.
• Sort mode Enrichment sorts have higher yield
  than purity sorts.
• The number of adjustment cells depends on the
  degree of difficulty of the flow cytometric
  measurement.

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Important definitions

• number of target cells in the sorted sample
• total number of particles in the sorted sample
• number of target cells in the sorted sample
• number of target cells as indicated by the sorter
• number of target cells in the sorted sample
• number of target cells in the original sample

Purity ()
Recovery ()
Yield ()
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• This makes the sort session more successful
• First inform us and discuss with us. The more
  we know the better for the experiment !
• If possible perform before sorting an
  analytical measurement at an analytical flow
  cytometer in order to check your staining.
• Provide as many positive and negative controls
  as you can, one set for each color to adjust the
  positive and negative population.
• Someone familiar with the samples needs to be
  reachable by phone during the sorting.

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• Discuss with the core staff
• Inform us about the intent of the experiment,
• Dye combination g suitable optics,
• Properties and preparation of the cells, cell
  number, necessary purity. g estimation of the
  sorting time.
• What you will do with the cells after sorting?
• Please contact us to schedule an appointment
  for cell sorting.
• Pathogens (L2) or genetically manipulated
  material (S2) ?
• Please complete the Cell Sorting Application
  Form which you will find in the internet
  http//www.cellsorter.de.
• The current sign up time for sorting is about
  1 week.
• Inform us as soon as possible about a
  Cancellation.

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Access The service is available by
appointment. The MoFlo is operated by the MoFlo
Specialists Karin Nispel and Joachim Ellwart.
Users bring prepared samples and the flow
cytometry specialists will sort them. To prevent
mismanagement of the instrument by many different
users the MoFlo needs to be handled only by
us.
The Cell Sorting Facility of the IMI-Institute
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Distribution of users of the sorting facility in
2005 total 144 sort sessions 27 sort projects
Bio-Companies 2
University Hospital Rechts der Isar 1
IMI 17
MPI of Neurobiology 25
Junior Research Groups 5
LMU Biozentrum 5
KMOLBI 23
University Hospitals LMU 4
GSF-Institutes Neuherberg 5
Dept. of Gene Expression 1
Clinical Cooperation Groups 12
GSF 63
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Last but not least

• Feedback
• about the outcome of your experiment,
  especially if things did not turn as expected, is
  essential for us. Please let us know and discuss
  alternative solutions with us.

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Contact persons Karin Nispel Joachim
Ellwart Contact information www.cell-sorter
.de
The Cell Sorting Facility of the Institute of
Molecular Immunology at the Helmholtz Center for
Environment and Health