Cell separation simplified: The IMag experience.

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Cell separation simplifiedThe IMag experience.
Jurg Rohrer, Ph.D.
BD Biosciences Pharmingen
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Presentation Outline

• Introduction
• Cell separation techniques
• Magnetic cell separations
• Examples of positive cell selections
• Mouse B220 cell selection
• Mouse CD8 cell selection
• Mouse Thy1.2 cell selection
• Examples of cell depletions
• Mouse B220 cell enrichment
• Mouse CD4 / T cell enrichment
• Mouse CD4-/CD8- T cell progenitor enrichment

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Cell Separation Techniques
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Cell Separation Using Magnetic Particles

• Magnetic particles offer a simple solution for
  the rapid and efficient isolation of pure cell
  populations.
• Magnetic particles can be used for the positive
  or negative selection of leukocyte
  subpopulations.
• Principal
• The target population is labeled with magnetic
  particles.
• Once labeled, the cells are placed within a
  magnetic field.
• Labeled cells are retained within the field and
  unlabeled cells are washed out.

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Cell Separation Using Magnetic Particles

• Two major magnetic cell separation technologies
  are currently available.
• Large magnetic particles ( 1200 nm)
• Small particles (50 nm)
• Both can give good recovery and purity.
• Each technology has its advantages and
  disadvantages.
• Advantages and disadvantages are a consequence
  of
• Particle size
• Magnetic field gradient required for the
  separation

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Large Magnetic Particles

• Advantages-
• Technically simple, separation done in test tubes
• Easy to scale-up the numbers of cells
• Requires a weaker magnet to remove labeled cells
• Disadvantages-
• May affect biology of cells in culture
  post-separation
• Cell/bead aggregates can lead to nozzle clogs on
  the flow cytometer

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Small Magnetic Particles

• Advantages-
• Gentle on cells, easily cultured post separation
• Compatible with flow cytometry
• Forward and side scatter unaffected on labeled
  cells
• Disadvantages-
• Requires a stronger magnet to remove labeled
  cells
• Requires special cell separation devices (columns
  and/or magnets)
• Scale up requires additional and/or larger
  columns
• Different magnets required for different columns

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Intermediate Size Magnetic Particles200nm IMag
particles

• Advantages-
• Technically simple, separation done in test tubes
• Easy to scale-up the numbers of cells
• Gentle on cells, can be cultured post separation
• Compatible with flow cytometry
• Forward scatter unaffected on labeled cells
• Side scatter increased on labeled cells
• Disadvantages-
• Requires a stronger magnet to remove labeled cells

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The IMag Cell Separation Protocol
3. Place tube on IMagnet, wait 6 minutes
... Stained cells migrate toward magnet.
1. Add antibody coated particles to cell
suspension.
2. Stain for 30 minutes, particles bind to
target population.
4. Remove supernatant which contains unstained
cells. Wash captured cells 2X.
5. For negative selections, analyze the
supernatant. For positive selections remove tube
from the IMagnet and analyze the captured cells.
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A Simple and Versatile Magnet
BD IMagnet

• Capacity
• 6- 12x75 tubes
• 1-12x107 cells/tube
• 7.2x108 cells total
• 2- 17x100 tubes
• 3-40 x107 cells/tube
• 8 x108 cells total

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Presentation Outline

• Introduction
• Cell separation techniques
• Magnetic cell separations
• Examples of positive cell selections
• Mouse B220 cell selection
• Mouse CD8 cell selection
• Mouse Thy1.2 cell selection
• Examples of cell depletions
• Mouse B220 cell enrichment
• Mouse CD4 T cell enrichment
• Mouse CD4-/CD8- T cell progenitor enrichment

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Cell Analysis by Flow Cytometry
1st
3rd
2nd
Side Scatter
CD19-PE
Propidium Iodide
55
92
Forward Scatter
Forward Scatter
B220-FITC
Cell Yield of Cells in wanted Fraction X
100 of cells expected from
the starting population
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Mouse CD8 Cell Selection Purity of Negative and
Positive Fractions
Fresh Splenocytes IMagnet
Magnetic Separation Column
IMag-DM particles IMag-MSC particles
Competitor
2
2
1
9
Negative Fractions
CD8
CD3
97
92
88
Positive Fractions
CD8
CD3
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Mouse CD8 Cell Selection Effect of particles on
FSC/SSC profile
Fresh Splenocytes IMagnet
Magnetic Separation Column IMag-DM
particles IMag-MSC particles
Competitor
SSC
FSC
77 Live 75 Live
64 Live 59 Live
Started With 20x106 cells per
condition, expected 1.8x106
Cell Count 1.1 1.2
1.3 Cell
Yield 59 61
64
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Mouse CD8 Cell Selection Scale Up From 10 to 70
Million Cells
The yields for all conditions were 60 5.
unwanted cells in each fraction
X106 cells used in each separation
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Mouse B220 Cell SelectionOutline

• Isolate B220 cells using
• (a) IMag-DM B220 particles
• (b) IMag-MSC B220 particles
• (c) Competitors B220 particles
• Flow cytometric analysis of negative and positive
  fractions
• Stimulate B220 cells with LPS
• Look at CD69 expression by flow cytometry
• Stimulate B220 cells with anti-mu
• Look at intracellular Calcium flux by
  fluorimeter

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Mouse B220 Cell SelectionPurity of Negative and
Positive Fractions
Fresh Splenocytes IMagnet
Magnetic Separation Column IMag-DM
particles IMag-MSC particles Competitor
Negative Fractions
60
4
2
2
CD19
B220
Positive Fractions
99
96
94
CD19
B220
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Mouse B220 Cell Selection Effect of particles
on FSC/SSC profile
Fresh Splenocytes IMagnet
Magnetic Separation Column
IMag-DM particles IMag-MSC particles
Competitor
SSC
FSC
75 Live 80 Live
81 Live 79 Live
Started With 20x106 cells per
condition, expected 12x106
Cell Count 7.8 6.7
7.0 Cell
Yield 64 54
55
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Mouse B220 Cell Selection24 Hour LPS Stimulation
Positively selected cells were stimulated for 24
hours with 20ug/ml LPS
Total Splenocytes IMagnet
Magnetic Separation Column IMag-DM
particles IMag-MSC particles
Competitor
80
78
81
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CD69
Non-stimulated Cells Stimulated Cells
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Mouse B220 Cell SelectionStimulation With
Anti-IgM
Calcium Flux Assay 1. Positively selected cells
washed in Ca2 free buffer. 2. Cells loaded with
INDO-1 AM 3. Cells stimulated with anti-mu Fab
20ug/ml. 4. Ca2 release measured for 120
seconds using a fluorometer.
Fresh Splenocytes IMag-DM particles IMag-MSC
particles
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Mouse Thy1.2 Cell SelectionOutline

• Isolate Thy1.2 cells using
• (a) IMag-DM Thy1.2 particles
• (b) IMag-MSC Thy1.2 particles
• Flow cytometric analysis of negative and positive
  fractions
• Stimulate Thy1.2 cells with plate bound anti-CD3
  soluble anti-CD28
• (a) look at CD69 expression by flow
  cytometry
• (b) look at IL2 production by ELISPOT

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Mouse Thy1.2 Cell Selection Purity of Negative
and Positive Fractions
Fresh Splenocytes
IMag-DM particles IMag-MSC
particles
4
4
Negative Fraction
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92
94
Thy1.2
Positive Fraction
CD3
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Mouse Thy1.2 Cell Selection Effect of particles
on FSC/SSC profile
Fresh Splenocytes IMag-DM particles
IMag-MSC particles
SSC
FSC
72 Live 85 Live
79 Live Started With 50x106 cells per
condition, expected 13x106
Cell Count 8.6 8.3
Cell Yield 66
64
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Mouse Thy1.2 Cell Selection 24 Hour Anti-CD3
Stimulation - CD69 Expression
Fresh Splenocytes
IMag-DM particles IMag-MSC
particles
92
86
77
CD69
Non-stimulated Cells Stimulated Cells
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Introduction to ELISPOT

• Very similar to an ELISA, except
• Add live cells to microwells (96 well plate),
  NOT supernatants, serum, plasma, or lysates.
• Purified or unpurified cells are activated in the
  microwells.
• Results are colored spots, each reactive cell
  producing the cytokine of interest leaves a mark
  in the microwell.

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ELISPOT
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Mouse Thy1.2 Cell Selection 24 Hour Anti-CD3
Stimulation - ELISPOT
Fresh Splenocytes Thy1.2 cells using
IMag-DM Particles Thy1.2 cells using
IMag-MSC particles
Fresh DM MSC
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Pointers For Tissue Culture
The positive selection process can affect cell
viability and performance in culture post
separation. To maximize the performance of the
selected cells in culture, keep these pointers in
mind

• Isolate cells in media rather than in PBS buffer
• A richer media like DMEM is better than RPMI
• Work quickly, get the cells into culture as soon
  as possible

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Presentation Outline

• Introduction
• Cell separation techniques
• Magnetic cell separations
• Examples of positive cell selections
• Mouse B220 cell selection
• Mouse CD8 cell selection
• Mouse Thy1.2 cell selection
• Examples of cell depletions
• Mouse B220 cell enrichment
• Mouse CD4 T cell enrichment
• Mouse CD4-/CD8- T cell progenitor enrichment

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B220 Cell SelectionComparing Positive Selection
and Depletion

• Isolate B220 cells by
• 1) Positive selection using B220 IMag particles
  with the IMagnet and a separation column.
• 2) Depletion using a depletion cocktail of CD4,
  Ter119 and CD43 with the IMagnet and a separation
  column.
• Place cells in culture for 6 hours with or
  without LPS.
• Examine CD25 expression on cells from each of the
  four conditions.
• Can one see upregulation of CD25 on unstimulated
  cells?

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B220 Cell SelectionComparing Positive Selection
and Depletion
Unmanipulated
60
CD19
B220
Yields IMagnet 63 Column 74 IMagnet -
60 Column - 36
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B220 Cell SelectionComparing Positive Selection
and Depletion
Non-stimulated LPS Stimulated
Unmanipulated
IMagnet Separation
Column Separation
7
15
8
19
CD25
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Mouse CD4 T Cell Isolation By DepletionOutline

• Isolate untouched CD4 T cells using a CD4
  enrichment cocktail (CD45R/B220, CD8, Ter 119,
  DX5, CD11b).
• CD4 enriched cells are placed in culture with
  Th1/Th2 polarizing cytokines.
• Analyze the supernatants produced by the Th1 and
  Th2 cultures for expression of relevant cytokines
  using the mouse Th1/Th2 CBA kit.
• Analyze the Th1 and Th2 cultured cells for
  transcripts of relevant cytokines using the mCK1
  RPA kit.

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Mouse CD4 T Cell Isolation By DepletionAnalysis
of Depleted Fractions
IMag-DM particles IMag-MSC particles

Fresh Splenocytes
94
98
24
CD4
CD3
Started off with 50 million total splenocytes per
condition The IMag-DM cell count was 10.3
million or a yield of 81 The IMag-MSC cell
count was 5.4 million cells or a yield of 44
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Mouse CD4 T Cell Isolation By DepletionSetting
Up Th1/Th2 Cultures

• CD4 enriched T cells
  put in culture
• In an ?CD3 coated well
  In an ?CD3 coated well
• with IL12, IFN?, anti-IL4 with IL4, IL2,
  anti-IFN?
• Culture
  cells for 5-6 days
• Re-stimulate ?CD3 ?
  CD28 for 4 hours
• Analyze using CBA
  and RPA kits

Th1 Polarization
Th2 Polarization
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Introduction to CBAThe Cytometric Bead Array

• This technology is based on the ELISA principle.
• Defined as a multiplexed fluorescent immunoassay
  for the flow cytometer
• -Designed to detect multiple analytes
  simultaneously in a single sample.
• -Saves many-fold on sample requirements compared
  to an ELISA.
• -A larger assay range requires fewer dilutions.

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Cytometric Bead Array
IFN-g
IL-4
IL-2


IL-5
TNF-a
IL-10
Wash
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Cytometric Bead ArrayAnalysis of cytokines in
the supernatant
MSC
DM
pg/ml
40000
30000
Th1
20000
10000
40000
30000
Th2
20000
10000
TNF? IFN? IL2
IL4 IL5
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Introduction to RPAThe Ribonuclease Protection
Assay
Probe (P32, Biotin)
mRNA source
Hybridization in Solution (stringent)
Ribonuclease
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Mouse CD4 T Cell Isolation By Depletion RPA
Analysis of Th1/Th2 Cells
Th1 or Th2 polarized T cells are harvested after
re-stimulation. The RNA is extracted. Purified
RNA is subjected to RPA analysis using the mCK1
probe set.
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Enrichment for Notch1 Cells in Mouse Thymus
Notch1 is expressed on immature CD4-CD8-
thymocytes. CD4-CD8- thymocytes make up 10 of
all thymocytes. This makes it difficult to see
Notch1 staining on total thymocytes. Therefore
remove the CD4CD8 thymocytes using a CD4/CD8
IMag depletion cocktail in conjunction with the
IMagnet.
Started with 150x106 total cells. Expected
15x106 and got 9.4x106. Therefore yield was 61
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mNotch1 analysis of Thymocytes
Unmanipulated and CD4CD8 depleted thymocytes
were fixed and permeabilized and then stained
with IM7 (CD44) and mN1a (Notch1) and analyzed by
flow cytometry.
Unmanipulated
Depleted
CD44
mN1a (Notch1)
Isotype Control
Isotype Control
mN1a (Notch1)
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Summary
IMag technology offers researchers a fast, high
performance, economical magnetic cell separation
system. The IMagnet is easy to use Works
with standard test tubes. Easy to
scale-up. Simple 15 minute separation protocols.
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Summary Continued
High Performance Consistently in positive fractions Excellent recovery for
positive selection protocols Excellent recovery
for depletion protocols Cost Effective Uses
inexpensive multiple capacity magnets No single
use magnetic separation columns
required Increased Flexibility Particles work
with the IMagnet Direct Magnet. Particles work
with Magnetic Separation Columns.
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Contributors
IMagnet Development RPA Analysis Alejandro
Uribe Benninghoff Don Jack DM and MSC
applications Cytokine Analysis Alejandro Uribe
Benninghoff Stephanie Widmann Jennifer
Bickel Homero Sepulveda Andrea Nguyen
Heather Baumhover Tiffany Clarke Ravi
Hingorani IMag Particles Carl Skold (Skold
Technologies) Ken Davis (BD Biosciences
Immunocytometry Systems) Majid Mehrpouyan (BD
Biosciences Immunocytometry Systems) Wing Liu