Gérard LIZARD - Inserm
EA7270 - Equipe ‘Biochimie du Peroxysome, inflammation et Métabolisme Lipidique’
Faculté des Sciences Gabriel
6, Bd Gabriel, 21000 Dijon - FRANCE
Caractérisation et méthodes d’études de la mort cellulaire
par cytométrie en flux
Université de Bourgogne
Gérard Lizard
Rabat, 29 novembre 2018
CytoMagh 2018
Two pathways of cell death leading to necrosis and apoptosis. At the top is shown a normal cell.
1A: Swelling. 1B: Vacuolization, blebbing, and increased permeability. 1C: Necrotic changes. ie,
coagulation, shrinkage, and karyolysis. 2A: Shrinkage and pyknosis. 2B: Budding and karyorhexis.
2C: Necrotic changes, ie, breakup into a cluster of apoptotic bodies.
Majno G & Joris I Am J Pathol 1995, 146: 3 - 15. Gérard Lizard,
ORGANELLES INVOLVED IN CELL DEATH
Organelles Apoptosis Autophagy Necrosis /
Necroptosis
- Mitochondria + +
(mitophagy) +
- Lysosomes + / - + + / -
- Endoplasmic-
reticulum (ER) + / - +
+ / -
- peroxisome ? +
(pexophagy)
?
Bröker LE et al. Clin Cancer Res 2005, 11: 3155-3162. Gérard Lizard,
4
Relations peroxysome / mitochondrie
Implication dans le contrôle de l’équilibre RedOx et l’activation de la mort cellulaire
Lismont C, Nordgren M, Van Veldhoven PP, Fransen M. Front Cell Dev Biol. 2015, 27;3:35.
Cell Death (initial concept)
Apoptosis apoptotic morphology
Active programmed cell death
Caspase-dependent cell death
• Mitochondrial pathway
(Associated or not with reticulum stress)
• Death receptor pathway
Necrosis necrotic morphology
Passive unprogrammed cell death
Classical /Canonical Necrosis
Cell Death Independent of Caspases
Kitanaka C & Kuchino Y Cell Death Differ 1999, 6: 508-515. Gérard Lizard,
Cell death
Apoptosis Autophagy Necrosis
Active programm cell death
Caspase-dependent
Type-1 cell death
Active programm cell death
Caspase-independent
Type-2 cell death
Active programm cell death
Caspase-independent cell death
Necroptosis (RIP1, RIP3)
Passive programm cell death
Caspase-independent cell death
Classical necrosis (primary necrosis)
Type-3 cell death
Secondary necrosis
Gérard Lizard,
AVAILABLE METHODS ALLOWING THE CHARACTERIZATION
OF APOPTOSIS, NECROSIS / NECROPOTOSIS, AND AUTOPHAGY
Gérard Lizard,
FUNCTIONNAL CRITERIA ASSOCIATED WITH APOPTOSIS,
NECROSIS, AND NECROPTOSIS
• Microscopy, flow cytometry, biochemistry
- enhanced permeability of cytoplasmic membrane (trypan blue, fluorescent probes, LDH)
- externalization of phosphatidylserine
double staining with AnnexinV /propidium iodide (PI) (or aminoactinomycine D (AAD))
to distinguish between normal (AnnexinV-/PI -), necrotic (AnnexinV+/PI+),
and apoptotic (AnnexinV+/PI-) cells
- Coloration SYTO16 - IP
- Sub-G1 peak (not present in normal and necrotic cells)
- loss of transmembrane mitochondrial potential: numerous fluorescent probes available.
Currently, DioC6(3) and JC1 are the most reliable.
- FLICA (fluorochromes labeled inhibitors of caspases): in situ identification of activated
caspases (does not permit to distinguish between necrosis and caspase-independent cell death)
- TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling): in situ identification
of internucleosomal DNA fragmentation
Gérard Lizard,
Electron microscopy A: Control. B: Cells treated by VP-16; apoptotic cells with condensed and perinuclear
chromatin are observed, cytoplasmic and nuclear membrane integrity are preserved as well as morphology of
mitochondria (arrow). C: Cells treated by NaN3; necrotic cells are characterized by a loss of integrity of
cytoplasmic and nuclear membranes, degradation of cytoplasm and chromatin.
Fluorescence microscopy after staining with Hoechst 33342 D: Control cells. Nuclei show regular contours. E:
Cells treated by VP-16; cell with fragmented (f) nucleus. F: Cells treated by VP-16; cell with condensed (c)
nucleus. G: Necrotic (n) cells observed under treatment by NaN3;
the nucleus is diffuse and irregular. (Lizard G et al. Cytometry 1995, 21: 275-283)
APOPTOSIS / NECROSIS: MORPHOLOGICAL CRITERIA
Gérard Lizard,
MORPHOLOGICAL CRITERIA ASSOCIATED WITH APOPTOSIS,
NECROSIS AND NECROPTOSIS
* Phase contrast microscopy: loss of refringence; loss or not of cell
adhesion?
* Brightfield microscopy: after staining with GIEMSA,…
* Fluorescence microscopy: Hoechst staining allows to easily distinguish
between normal, necrotic and apoptotic cells (nuclear criteria)
* Transmission electron microscopy (nuclear criteria)
* Flow cytometry: changes of light scatter properties on non-fixed cells
(FSC ↓ ; SSC more or less ↑ )
Control 7-keto
40 µg/ml
U937 G
Gérard Lizard,
http://www.amnis.com
Henery S et al. Apoptosis 2008, 13: 1054-1063
Annexin V-FITC / PI (Amnis technology)
Gérard Lizard,
Phosphatidylserine externalization : Annexin V-FITC / PI test
Apoptosis
Necrosis
(phosphatidylserines)
V A
N
Annexin V - FITC
PI (o
r 7A
AD
)
Control Treated
PI: propidium iodide
7 AAD: 7 amino actinomycin D
Viable (V)
Apoptotic (A)
Necrotic (N)
Annexine V PI Cells
Gérard Lizard,
Towards an Understanding of Apoptosis Detection by SYTO Dyes Donald Wlodkowic,1* Joanna Skommer,1 and Jukka Pelkonen1,2
1Institute of Clinical Sciences, Department of Clinical Microbiology, University of Kuopio, Kuopio, Finland
2Department of Clinical Microbiology, Kuopio University Hospital, Kuopio, Finland
Cytometry Part A 71A:61–72 (2007)
Necrotic cells
Viable cells Apoptotic cells
Gérard Lizard,
Nu
mb
er
of
ce
lls
Sub-G1
control
cholesterol 7ß-hydroxycholesterol
7-ketocholesterol
HUVECs
Lizard G et al. Am J Pathol 1996, 148: 1625-1638
Sub-G1 (FCM)
apoptosis
primary or secondary necrosis
Gérard Lizard,
0
20
40
60
80
100
0 6 12 18 24
Time of treatment (hours) % c
ell
s w
ith
dep
ola
rize
d m
ito
ch
on
dri
a
control
7-ketocholesterol (40 µg/ml)
7b-hydroxycholesterol (20 µg/ml)
Control 7-ketocholesterol 7-hydroxycholesterol
Mitochondrial potential (FCM)
Gérard Lizard,
Grabarek J & Darzynkiewicz Z Exp Hematol 2002, 30: 982-989
FLICA / FLISP (Microscopy, Flow Cytometry)
Gérard Lizard,
TUNEL (Flow Cytometry)
Gérard Lizard,
TUNEL (Amnis technology)
Gérard Lizard,
Lizard G et al., Arterioscler Thromb Vasc Biol 1999, 19: 1190-2000
Phase contrast microscopy, Hoechst 33342, GIEMSA, TUNEL
Apoptosis = TUNEL positive cells
Necrosis = TUNEL negative cells
Gérard Lizard,
BIOCHEMICAL CRITERIA ASSOCIATED WITH
APOPTOSIS, NECROSIS, AND NECROPTOSIS
* Analysis of the DNA fragmentation pattern on agarose gel
- Apoptosis: DNA ladder, multiple of 150-200 base pairs
(internucleosomal DNA fragmentation)
- Necrosis: no DNA ladder, smears
* Enzymatic activities (LDH)
* Electrophoresis on acrylamide gel and Western Blotting
Gérard Lizard,
Huang WC, Chen CL, Lin YS, Lin CF. Apoptotic sphingolipid ceramide in cancer
therapy.
J Lipids. 2011;2011:565316.
Anticorps anti-céramides
Hoechst
Cellule
normale
Cellule
pré-apoptotique
Cellule
apoptotique
Exp
res
sio
n d
e c
éra
mid
es
Stimuli apoptotique : génération de céramides
(détection par immunofluorescence)
MCF-7 MCF-7/c3
Prunet C et al. Cell Death Differ, 2001; J Biochem Mol Toxicol, 2005
Caspase-3/7
Caspase-8
Caspase-6
DYm without caspase-3
Oncosis
AIF, EndoG
cytochrome-C
Caspase-9
Caspase-3
Caspase-7 Caspase-8
Bid
t-Bid
Apoptosis
PARP
ICAD CAD
Necrosis/Oncosis = no caspase activation
Gérard Lizard,
Gérard Lizard,
CHARACTERIZATION OF AUTOPHAGY:
‘VISUAL’ CRITERIA
7KC (50 µM, 24 h)
Control
EtOH (0.1%)
0.5 µm
1 µm
1 µm
1 µm
1 µm
ap
ap
ap: autophagosome; apl: autophagolysosome
1 µm
apl
0.1 µm
A
B
C
D
E
F
G
H apl
0.1 µm
Gérard Lizard
CHARACTERIZATION OF AUTOPHAGY:
ULTRASTRUCTURAL CRITERIA – Transmission Electron Microscopy
CHARACTERIZATION OF AUTOPHAGY: BIOCHEMICAL CRITERIA Murine oligodendrocytes (158N) - autophagy and apoptosis (oxiapoptophagy)
Co
ntr
ol
EtO
H (
1%
)
α-c
yclo
dex
trin
(12
5 µ
M)
α-t
oco
ph
ero
l (4
00
µM
)
DH
A (
50
µM
)
7K
C (
50
µM
)
7K
C +
α-t
oco
7K
C +
DH
A
7K
C +α
-to
co+
DH
A
7β
-OH
C (
50
µM
)
7β
-OH
C +α
-to
co
7β
-OH
C +
DH
A
7β
-OH
C +
α-t
oco
+DH
A
24
S-O
HC
(5
0µ
M)
24
S-O
HC
+α
-to
co
24
S-O
HC
+D
HA
24
S-O
HC
+ α
-to
co +
DH
A
Caspase -3 (35 kDa)
Cleaved Caspase -3 (17 kDa)
PARP (116 kDa)
Cleaved PARP (89 kDa)
β-Actin (42 kDa)
LC3-I (18 kDa)
LC3-II (16 kDa)
Bcl-2 (26 kDa)
Ratio LC3-II/LC3-I 0.6 0.5 3.8 0.8 0.6 0.7 1.7 1.3 1.2 1.3 3.4 1.7 1.2 0.9
EtO
H (
0.1
%)
EtO
H (
1.1
%)
α-c
yclo
dex
trin
+ E
tOH
(0
.1 %
)
α-c
yclo
dex
trin
+ E
tOH
(1
.1 %
)
0.6 0.6 0.6 0.6 0.7 0.5 0.4
Gérard Lizard,
Apoptosis Necrosis Necroptosis
Phosphatidyl serine
externalization
(Annexin V positive + PI positive)
Mitochondrial depolarization
Random DNA degradation
RIP1 (and RIP3)
expression
Phosphatidyl serine
externalization
(Annexin V positive + PI negative)
Mitochondrial depolarization
Non Random DNA degradation
(DNA ladder)
Caspases activation
Gérard Lizard,
Autophagy
Oxiapoptophagy
Necroapoptophagy
LC3 conversion:
LC3I to LC3-II
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