Measurement of cell proliferation by dye dilution

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Measurement of cell proliferation by dye dilution

• Dr Adrian RobinsImmunology, Queens Medical
  Centre, Nottingham

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Divisions 0 1 2 3
Fluorescence 1 1/2 1/4 1/8
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Dyes available for dilution proliferation method

• CFSE
• Lyons AB, Parish CR. Determination of Lymphocyte
  Division by Flow-Cytometry. Journal of
  Immunological Methods 1994 171 131-37.
• PKH26
• Givan AL, Fisher JL, Waugh M, Ernstoff MS,
  Wallace PK. A flow cytometric method to estimate
  the precursor frequencies of cells proliferating
  in response to specific antigens. Journal of
  Immunological Methods 1999 230 99-112.

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CFSE Intracellular Labelling
O
O
O-Ac
Ac-O
O
HO
intracellular esterase
intracellular esterase
COOH
COOH
O
OC-O-N
OC
N-H
O
Carboxy-fluorescein-diacetate- succinimidal ester
(CFDA-SE or CFSE) (non-fluorescent, membrane
permeable)
covalent link to intracellular protein
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CFSE labelling method

• CFSE (Molecular Probes) 5mM stock in dry DMSO
• Cells for labelling in 2ml serum free RPMI
  medium
• Add 4ul CFSE to 2ml RPMI, then immediately
• mix thoroughly with cells
• Incubate for 10min room temperature
• Wash twice in RPMIserum
• note that incubation at 37deg for several hours
  is required for fluorescence from non covalently
  bound carboxy-fluorescein to be lost from cells

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PHA stimulation
3
4
2
5
0
1
6
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Fluorescence spillover from fluorescein
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Unstimulated T cells
CD45 RO (Memory cells)
CD45 RA (naïve T cells)
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Stimulation with PHA
CD45 RO (Memory cells)
CD45 RA (naïve T cells)
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CD45 RA
CD45 RO
Unstimulated PHA
stimulated
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Quantitative assessment of cell proliferation

• measure the number of cells in each peak
• but -
• we measure a set total number of cells, so as
  cells divide, the dividing populations dilute
  the non-dividing cells.

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Simulation of cell division

• 100
• 50 100
• 50 25 150
• 50 25 50 200
• 50 25 50 100 200
• 50 25 50 100 175 50 450 cells
• 4.5x
• 11.1 5.6 11.1 22.2 38.9 11.1 100

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Quantitative assessment of cell proliferation

• If you want to calculate parameters of cell
  proliferation, you need to estimate the number of
  cells from which the proliferating population
  originated.
• Estimate the percentage of cells in each peak
• Divide the percentage in each peak by 2n, where
  n is the number of divisions for that peak
• 0 divisions - divide by 1
• 1 divisions - divide by 2
• 2 divisions - divide by 4
• 3 divisions - divide by 8
• 4 divisions - divide by 16
• etc
• Add up the resulting percentages - the total is
  a measure of the number of cells from which the
  cells originated

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Simulation of cell division

• 100
• 25 50 100 175 50 450 cells
• 
  4.5x
• 11.1 5.6 11.1 22.2 38.9 11.1 100
• /1 /2 /4 /8 /16 /32
• 11.1 2.8 2.8 2.8 2.4 0.35 22.25
• 22.25 -gt 100
• 100/22.25 4.49x expansion

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Spreadsheet to make calculations
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Parameters of proliferation

• Total expansion - good for when most cells
  proliferate
• Proportion of cells proliferating - important
  for antigen or superantigen stimulation
• Degree of expansion of proliferating cells -
  sensitive measure of proliferation when only a
  few cells are proliferating

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Simulation of cell division antigen specific

• 100
• 98 4
• 98 0 8
• 98 0 2 12
• 98 0 2 4 16
• 98 0 2 4 12 8 126 cells
• 79 0 1.6 3.2 9.7 6.4 100

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Comparison of incubation conditions for cfse
labelling

• superantigen stimulation
• Labelling total exp exp prolif cells
• room temp 3.30 6.54
• 3.33 6.61
• 3.22 6.38
• 3.11 6.90
• 37deg 3.09 6.25
• 3.13 6.33
• 3.08 6.25
• 3.04 6.28
• p0.02

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Example of curve fitting from Givan et al
1999. Cells stained with PKH26 curve fit using
MODFIT
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Curve fitting using WEASEL
http//www.wehi.edu.au/cytometry/WEASEL.html
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Combination of intracellular and cell surface
labels with cfse
IFN gamma producing cells gated
Stimulation with staph enterotoxin B over 7 days,
then 4h restimulation with brefeldin A
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Summary

• The dye dilution method is a powerful way to
  monitor lymphocyte division
• the phenotype of dividing cells can be
  identified
• the dependence of cell functions on division can
  be directly determined with appropriate markers
• cell proliferation in vivo can be measured
• does not depend on radioactive isotopes, and
  does not require critical choice of pulse time
• may be suitable for measuring mitogen,
  superantigen and antigen stimulated proliferation

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Measurement of cellular cytotoxicity

• Gold standard
• Chromium release assay
• target cells labelled with radioactive chromate,
• incubated with effector cells,
• supernatant sampled and radio isotope counted
• Alternatives
• Target cell viability using membrane probes
• Effector cell function by detecting granule
  exocytosis

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Membrane viability probes

• measure target cell viability with membrane
  probes
• topro 3 - permeability probe
  (red excitation, red
  fluorescence)
• Annexin V fitc - membrane lipid reorientation
  probe (blue excitation, green fluorescence)
• label target cells to distinguish them from
  effector cells
• pkh26 - membrane label
  (blue excitation,
  yellow fluorescence)

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Flow cytometric analysis of cellular
cytotoxicity K562 target cells
Analysis of target cell death using annexin V
and topro 3
Target cells gated using pkh26
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Flow cytometric analysis of cellular
cytotoxicity K562 target cells, peripheral
blood mononuclear cells as effectors
61 effector to target cells
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Flow cytometric analysis of cellular
cytotoxicity K562 target cells, peripheral
blood mononuclear cells as effectors
Effector target ratio
0 61 121
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Detection of granule exocytosis
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Exocytosis assay

• CD107 is an intracellular granule marker
• CD107 is surface expressed during the process of
  granule exocytosis
• Labelled CD107 antibody included in cytotoxicity
  assay

Unstimulated
SEB stimulated
M.R. Betts et al. / Journal of Immunological
Methods 74 281 (2003) 6578
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Inverse relation between CD107 and perforin
M.R. Betts et al. / Journal of Immunological
Methods 74 281 (2003) 6578
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Combination with tetramer staining
M.R. Betts et al. / Journal of Immunological
Methods 74 281 (2003) 6578
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Thanks to

• Dr Heather Judge
• Mian Chen
• Izaty Farhana
• Terence Buhong
• Julie Swales