References((

1(Tinari,(A.,(Giammarioli,(A.(M.,(Manganelli,(V.,(Ciarlo,(L.(&(Malorni,(W.(Analyzing(morphological(and(ultrastructural(features(in(cell(death.(Methods(Enzymol(442,(1B26((2008)(2(Sato,(T.(et(al.(Single(Lgr5(stem(cells(build(cryptBvillus(structures(in(vitro(without(a(mesenchymal(niche.(Nature(459,(262B265((2009).(3(University(of(Texas,(MD(Anderson(Cancer(Center,(Core(facility((web(8th((December,(2012)(4(Degterev,(A.(et(al.(Chemical(inhibitor(of(nonapoptoTc(cell(death(with(therapeuTc(potenTal(for(ischemic(brain(injury.(Nat(Chem(Biol(1,(112B119((2005).((

((

The(aim(of(this(project(was(to(unify( in(a(pathway(all(the(different( informaTon(about(necroptosis(to(to(give(some(light(both(on(how(necroptosis( is(driven(and(also(how(we(are(able(to(detect( it(on(Tssues( in(order( to(allow( further( invesTgaTons( take( in(consideraTon( this(pathway(as( it( can(be(of( special( interest( for( cancer( therapies.(This(was(performed( :( searching(at(Pubmed(for(necroptoTc(papers(laZer(the(literature(and(product(companies(for(different(techniques(that(allow(its(differenTaTon(

(

Conclusions(

((

Cell(survival(has(been(the(subject(of(acTve(research(for(several(years,(which(has(also(included(cell(death,(as(it(is(an(important(mechanism(for(Tssue(homeostasis.(Historically,(it(has(been(described(a(caspaseBdriven(programed(cell(death(named(as(apoptosis,(and(a(“non(programed(cell(death”,(necrosis.(However,(recent(studies(indicate(that(necrosis(can(also(be(driven(by(different( sTmulus( through( a( programed( pathway;( therefore( a( new( term( has( been( introduced:( necroptosis.( It( refers( to( a( cellBdeath( receptorBinduced,( caspase( independent,( highly(regulated(type(of(programed(cell(death(process(with(morphological(resemblance(to(necrosis.(Unfortunately,(whereas(apoptosis(has(been(largely(studied,(the(molecular(composiTon(of(the(necroptoTc(pathway(has(remained(elusive(unTl(now,(as(nowadays(it(is(sTll(hard(to(visually(disTnguish(it(

Author:(Ricard(GarciaBCarbonell,(Biomedical(Sciences(Degree,(University(Autonomus(of(Barcelona((Supervisor:(Mariona(Terradas(Ill,(Department(of(Cell(Biology,(Physiology(and(Immunology(of(the(University(Autonomus(of(Barcelona((

Apoptosis' Necroptosis'Membrane'bebbling'without'loss'of'membrane'integrity' Loss'of'membrane'integrity'Aggregation'and'fragmentation'of'the'chromatin' No'chromatin'fragmentation'Cytoplasm'condensation'and'nuclear'collapse'

Mitochondrial'and'cytoplasm'swallowing'

At'the'end,'cellular'small'bodies'are'formed' Ends'with'cellular'lysis'Apoptotic'bodies'formation' No'vesicles'are'formed'Mitochondrial'permeabilization'' Swallowing'induced'organelles'fision'Less'inflammation'associated'response' Inflammation'associated'response'Possible'activation'of'caspase'pathway''Loss'of'membrane'asymmetry'

Inhibition'of'caspases'is'required'

'

Electronic(Microscopy(Nowadays,(the(goldBstandard(way(to(disTnguish(between(apoptosis(and(

necroptosis(is(electronic(microscopy(

SCalibur flow fluorocytometer (Becton Dickinson)equipped with a water-cooled argon-ion laser at 488 nm.To clearly see the dead cells on the dot plot, PI-positivecells are gated, e.g. in red, in the FL3 histogram. The regionof analysis was gated (R1), and gating must be largeenough in order to measure both living and dying cellpopulations.

2.3. Transmission electron microscopy

Transmission electron microscopic (TEM) analysis hasbeen considered a ‘golden standard’ in cell death research.It provides two- and three-dimensional (using Fouriermethods) images of the inside of cells, which makes it pos-sible to understand biological structure–function relation-ships at cellular, subcellular and molecular levels [22,23].Although TEM is time consuming and requires moreexpensive equipment, it offers much higher resolving power(0.1–0.4 nm), thereby providing much more detailed infor-mation about cell morphology. TEM is considered themost accurate method for distinguishing apoptosis fromnecrosis in cell cultures. However, preparation of dyingcells for electron microscopy may be difficult because dyingcells typically detach from their substrate, and spinningdown these floating cells may cause damage to their origi-nal morphology. Below we suggest a method that helps tocircumvent this problem by using macrophages attached tothe bottom of tissue culture plates to capture dying apop-totic and necrotic cells. The samples were viewed with aJeol 1200 EXII TEM at an accelerating voltage of 80 kV.

2.3.1. Sample preparationMacrophages were seeded in adherent 6-well plates at

5 ! 105 cells per well and target cells in uncoated 6-well sus-pension plates at 5 ! 105 cells per well. The target cells weregrown in suspension plates so that the population of dyingcells could be transferred easily to the well containingattached macrophages. Apoptosis and necrosis wereinduced in target L929sAhFas cells either by agonistic

anti-Fas antibody (250 ng/ml, for at least 1 h) or mTNF(10,000 IU/ml, for at least 7 h), respectively. The choice oftime points depend on the kinetics of cell death and haveto be optimized for each cell line and death inducer. A cocul-ture of macrophages with target cells was established in a 1:1ratio and incubated for at least 1 h. After that, cocultures ofmacrophages and target cells were fixed overnight at 4 !C inthe 6-well plate by immersion in TEM fixation buffer (2%glutaraldehyde containing 1 mM CaCl2 and 0.1 M sucrosebuffered with 0.1 MNa-cacodylate, pH 7.4). Following sev-eral rinses in 100 mM Na-cacodylate containing 7.5% w/vsucrose, the samples were post-fixed overnight at 4 !C in1%w/vOsO4 in the same buffer (without sucrose). Theywerethen washed several times in 100 mM Na-cacodylate con-taining 7.5% w/v sucrose and dehydrated in a graded seriesof ethanol (50% for 15 min, 70% for 20 min, 90% for30 min and 100% for 90 min). Following infiltrationwith 100% ethanol, they were infiltrated with a mixtureof ethanol and LX-112 resin (Ladd Research Industries,USA) as follows: 1:1 for 30 min and 1:2 for 30 min. Itwas finally infiltrated with 100% resin for 120 min, andthen polymerized for at least 48 h at 60 !C. Finally, theplastic was broken off of the polymerized block, sawedinto pieces to fit the ultramicrotome holders, and ultrathin sections of 60 nm were mounted on formvar-coated100-mesh copper grids. The samples were evaporated ina JEOL (JEC-530) autocarbocoater at 4 V for 3 s andthen stained with uranyl acetate (7.5% in bi-distilledwater, 1 drop per grid for 20 min) and Reynold’s leadcitrate (1 drop per grid for 10 min). The semi-thin sec-tions were examined by TEM.

Two of the earliest classic ultrastructural changes detect-able in apoptosis are formation of uniformly dense massesof chromatin distributed against the nuclear envelope [24]and persistence of a nucleolar structure until the very latestages [25]. Apoptotic cells (Fig. 2B) are typically charac-terized by the loss of specialized surface structures, suchas microvilli and cell–cell contacts, condensation of cyto-plasm, and formation of membrane-bound apoptotic

Fig. 2. Analysis of cell morphology of apoptotic and necrotic cells by transmission electron microscopy. Unstimulated L929sAhFas fibrosarcoma cell (A)and cells exposed either to agonistic anti-Fas for 1 h (B) or to hTNF for 7 h (C). (A) The cell shows microvilli protruding from the entire surface, asmoothly outlined nucleus with chromatin in the form of heterochromatin and well-preserved cytoplasmic organelles. (B) Apoptotic cell with sharplydelineated masses of condensed chromatin, convolution of the cellular surface and formation of apoptotic bodies. Note the nucleolus (arrow head) near acup-shaped chromatin margination. (C) Necrotic cell near the macrophage (asterisk) with clumps of chromatin with ill-defined edges, swollenmitochondria and loss of plasma membrane integrity. Scale, bars: 1 lm.

D.V. Krysko et al. /Methods 44 (2008) 205–221 209

SCalibur flow fluorocytometer (Becton Dickinson)equipped with a water-cooled argon-ion laser at 488 nm.To clearly see the dead cells on the dot plot, PI-positivecells are gated, e.g. in red, in the FL3 histogram. The regionof analysis was gated (R1), and gating must be largeenough in order to measure both living and dying cellpopulations.

2.3. Transmission electron microscopy

Transmission electron microscopic (TEM) analysis hasbeen considered a ‘golden standard’ in cell death research.It provides two- and three-dimensional (using Fouriermethods) images of the inside of cells, which makes it pos-sible to understand biological structure–function relation-ships at cellular, subcellular and molecular levels [22,23].Although TEM is time consuming and requires moreexpensive equipment, it offers much higher resolving power(0.1–0.4 nm), thereby providing much more detailed infor-mation about cell morphology. TEM is considered themost accurate method for distinguishing apoptosis fromnecrosis in cell cultures. However, preparation of dyingcells for electron microscopy may be difficult because dyingcells typically detach from their substrate, and spinningdown these floating cells may cause damage to their origi-nal morphology. Below we suggest a method that helps tocircumvent this problem by using macrophages attached tothe bottom of tissue culture plates to capture dying apop-totic and necrotic cells. The samples were viewed with aJeol 1200 EXII TEM at an accelerating voltage of 80 kV.

2.3.1. Sample preparationMacrophages were seeded in adherent 6-well plates at

5 ! 105 cells per well and target cells in uncoated 6-well sus-pension plates at 5 ! 105 cells per well. The target cells weregrown in suspension plates so that the population of dyingcells could be transferred easily to the well containingattached macrophages. Apoptosis and necrosis wereinduced in target L929sAhFas cells either by agonistic

anti-Fas antibody (250 ng/ml, for at least 1 h) or mTNF(10,000 IU/ml, for at least 7 h), respectively. The choice oftime points depend on the kinetics of cell death and haveto be optimized for each cell line and death inducer. A cocul-ture of macrophages with target cells was established in a 1:1ratio and incubated for at least 1 h. After that, cocultures ofmacrophages and target cells were fixed overnight at 4 !C inthe 6-well plate by immersion in TEM fixation buffer (2%glutaraldehyde containing 1 mM CaCl2 and 0.1 M sucrosebuffered with 0.1 MNa-cacodylate, pH 7.4). Following sev-eral rinses in 100 mM Na-cacodylate containing 7.5% w/vsucrose, the samples were post-fixed overnight at 4 !C in1%w/vOsO4 in the same buffer (without sucrose). Theywerethen washed several times in 100 mM Na-cacodylate con-taining 7.5% w/v sucrose and dehydrated in a graded seriesof ethanol (50% for 15 min, 70% for 20 min, 90% for30 min and 100% for 90 min). Following infiltrationwith 100% ethanol, they were infiltrated with a mixtureof ethanol and LX-112 resin (Ladd Research Industries,USA) as follows: 1:1 for 30 min and 1:2 for 30 min. Itwas finally infiltrated with 100% resin for 120 min, andthen polymerized for at least 48 h at 60 !C. Finally, theplastic was broken off of the polymerized block, sawedinto pieces to fit the ultramicrotome holders, and ultrathin sections of 60 nm were mounted on formvar-coated100-mesh copper grids. The samples were evaporated ina JEOL (JEC-530) autocarbocoater at 4 V for 3 s andthen stained with uranyl acetate (7.5% in bi-distilledwater, 1 drop per grid for 20 min) and Reynold’s leadcitrate (1 drop per grid for 10 min). The semi-thin sec-tions were examined by TEM.

Two of the earliest classic ultrastructural changes detect-able in apoptosis are formation of uniformly dense massesof chromatin distributed against the nuclear envelope [24]and persistence of a nucleolar structure until the very latestages [25]. Apoptotic cells (Fig. 2B) are typically charac-terized by the loss of specialized surface structures, suchas microvilli and cell–cell contacts, condensation of cyto-plasm, and formation of membrane-bound apoptotic

Fig. 2. Analysis of cell morphology of apoptotic and necrotic cells by transmission electron microscopy. Unstimulated L929sAhFas fibrosarcoma cell (A)and cells exposed either to agonistic anti-Fas for 1 h (B) or to hTNF for 7 h (C). (A) The cell shows microvilli protruding from the entire surface, asmoothly outlined nucleus with chromatin in the form of heterochromatin and well-preserved cytoplasmic organelles. (B) Apoptotic cell with sharplydelineated masses of condensed chromatin, convolution of the cellular surface and formation of apoptotic bodies. Note the nucleolus (arrow head) near acup-shaped chromatin margination. (C) Necrotic cell near the macrophage (asterisk) with clumps of chromatin with ill-defined edges, swollenmitochondria and loss of plasma membrane integrity. Scale, bars: 1 lm.

D.V. Krysko et al. /Methods 44 (2008) 205–221 209

SCalibur flow fluorocytometer (Becton Dickinson)equipped with a water-cooled argon-ion laser at 488 nm.To clearly see the dead cells on the dot plot, PI-positivecells are gated, e.g. in red, in the FL3 histogram. The regionof analysis was gated (R1), and gating must be largeenough in order to measure both living and dying cellpopulations.

2.3. Transmission electron microscopy

Transmission electron microscopic (TEM) analysis hasbeen considered a ‘golden standard’ in cell death research.It provides two- and three-dimensional (using Fouriermethods) images of the inside of cells, which makes it pos-sible to understand biological structure–function relation-ships at cellular, subcellular and molecular levels [22,23].Although TEM is time consuming and requires moreexpensive equipment, it offers much higher resolving power(0.1–0.4 nm), thereby providing much more detailed infor-mation about cell morphology. TEM is considered themost accurate method for distinguishing apoptosis fromnecrosis in cell cultures. However, preparation of dyingcells for electron microscopy may be difficult because dyingcells typically detach from their substrate, and spinningdown these floating cells may cause damage to their origi-nal morphology. Below we suggest a method that helps tocircumvent this problem by using macrophages attached tothe bottom of tissue culture plates to capture dying apop-totic and necrotic cells. The samples were viewed with aJeol 1200 EXII TEM at an accelerating voltage of 80 kV.

2.3.1. Sample preparationMacrophages were seeded in adherent 6-well plates at

5 ! 105 cells per well and target cells in uncoated 6-well sus-pension plates at 5 ! 105 cells per well. The target cells weregrown in suspension plates so that the population of dyingcells could be transferred easily to the well containingattached macrophages. Apoptosis and necrosis wereinduced in target L929sAhFas cells either by agonistic

anti-Fas antibody (250 ng/ml, for at least 1 h) or mTNF(10,000 IU/ml, for at least 7 h), respectively. The choice oftime points depend on the kinetics of cell death and haveto be optimized for each cell line and death inducer. A cocul-ture of macrophages with target cells was established in a 1:1ratio and incubated for at least 1 h. After that, cocultures ofmacrophages and target cells were fixed overnight at 4 !C inthe 6-well plate by immersion in TEM fixation buffer (2%glutaraldehyde containing 1 mM CaCl2 and 0.1 M sucrosebuffered with 0.1 MNa-cacodylate, pH 7.4). Following sev-eral rinses in 100 mM Na-cacodylate containing 7.5% w/vsucrose, the samples were post-fixed overnight at 4 !C in1%w/vOsO4 in the same buffer (without sucrose). Theywerethen washed several times in 100 mM Na-cacodylate con-taining 7.5% w/v sucrose and dehydrated in a graded seriesof ethanol (50% for 15 min, 70% for 20 min, 90% for30 min and 100% for 90 min). Following infiltrationwith 100% ethanol, they were infiltrated with a mixtureof ethanol and LX-112 resin (Ladd Research Industries,USA) as follows: 1:1 for 30 min and 1:2 for 30 min. Itwas finally infiltrated with 100% resin for 120 min, andthen polymerized for at least 48 h at 60 !C. Finally, theplastic was broken off of the polymerized block, sawedinto pieces to fit the ultramicrotome holders, and ultrathin sections of 60 nm were mounted on formvar-coated100-mesh copper grids. The samples were evaporated ina JEOL (JEC-530) autocarbocoater at 4 V for 3 s andthen stained with uranyl acetate (7.5% in bi-distilledwater, 1 drop per grid for 20 min) and Reynold’s leadcitrate (1 drop per grid for 10 min). The semi-thin sec-tions were examined by TEM.

Two of the earliest classic ultrastructural changes detect-able in apoptosis are formation of uniformly dense massesof chromatin distributed against the nuclear envelope [24]and persistence of a nucleolar structure until the very latestages [25]. Apoptotic cells (Fig. 2B) are typically charac-terized by the loss of specialized surface structures, suchas microvilli and cell–cell contacts, condensation of cyto-plasm, and formation of membrane-bound apoptotic

Fig. 2. Analysis of cell morphology of apoptotic and necrotic cells by transmission electron microscopy. Unstimulated L929sAhFas fibrosarcoma cell (A)and cells exposed either to agonistic anti-Fas for 1 h (B) or to hTNF for 7 h (C). (A) The cell shows microvilli protruding from the entire surface, asmoothly outlined nucleus with chromatin in the form of heterochromatin and well-preserved cytoplasmic organelles. (B) Apoptotic cell with sharplydelineated masses of condensed chromatin, convolution of the cellular surface and formation of apoptotic bodies. Note the nucleolus (arrow head) near acup-shaped chromatin margination. (C) Necrotic cell near the macrophage (asterisk) with clumps of chromatin with ill-defined edges, swollenmitochondria and loss of plasma membrane integrity. Scale, bars: 1 lm.

D.V. Krysko et al. /Methods 44 (2008) 205–221 209

Electronic*density*changes*within*apoptotic*

cell1*

Healthy*cell1* Healthy*mitochondria1* Mitochondrial*swelling1*

Apoptotic*cell1* Necroptotic*cell1*

(Different(approaches(can(be(performed(in(order(to(disTnguish(between(them.(

(This(work(shows(a(novel(pathway(that(has(remained(unstudied(and(classified(as(a(“nonBregulated(cell(death”.(Weather(necrosis(is(just(triggered(by(the(aboveBmenToned(signaling(or(also(by(other(molecules(it(is(sTll(unknown(but(this(pathway(supports(the(idea(that(every(cell(outcome(is(due(to(a(complex(signaling(events(in(a(finally(balanced(but(sTll(unknown(state.((In(order(to(sum(up,(a(caspase(independent(program,(named(necroptosis,(in(which(RIP1,(RIP3(and(MLKL(play(an(important(role(can(also(trigger(cell(death.(Because(it(has(been(discovered(recently,(liZle(is(known(on(how(it(is(driven(however(further(invesTgaTon(should(be(done,(as(it(can(be(an(important(way(to(fight(cancer(because(it(usually(express(some(necroptoTc(iniTator(factors(that(just(need(a(push(up(to(prompt(those(specific(cancer(cells(towards(necroptosis.(This(work(also(shows(several(approaches(to(detect(necroptosis(in(order(to(be(taken(in(account(in(further(invesTgaTons(

How(to(disTnguish(between(apoptosis(and(necroptosis( Apoptosis(or(necroptosis?(

IntroducTon(

Aims(and(methodology(

(Pathway(created(by(Ricard(GarciaBCarbonell(under(

! Apoptois! Necroptosis!

Tunel! Clearly!positive!stained!cells! Pale!stained!cells!

Cleaved!caspases:! ! !3! Panapoptotic!marker!

Negative!cells!8! Extrinsic!pathway!marker!9! Intrinsic!pathway!marker!

Annexin!V!Positive!cells!due!to!

asymmetric!membrane!rupture!

Negative!cells!

Propidium!iodae(PI)! Negative!cells! Positive!nuclear!staining!Hematoxilina!i!eosina! Collapsed!cells! Not!distinguishable!

Electronic!microscopy! Major!changes!involving!mitochondria!and!nucleus!

!