Questo ha funzionato è stato sostenuto da sovvenzioni dal National Health e il Medical Research Council (GNT1125033 e GNT1140187) e Australian Research Council (DP170103790) a I.K.H.P.

1. induzione di apoptosi
<ol><li>Centrifugare il campione di cellule a 300 x g per 5 min ed eliminare il surnatante per rimuovere eventuali detriti cellulari pre-esistenti. Nota: Quando si utilizza cellule aderenti, cellule del seme in anticipo e lavare con soluzione tampone fosfato (PBS) prima di induzione di apoptosi: 1x.</li>
	<li>Determinare il numero di cell e raccogliere le cellule. Nota: A seconda post-dell'isolamento di test, si consiglia un numero di cellulare iniziale di almeno 1 x 107 cellule.</li>
	<li>Risospendere in completa per i media (mezzo rispettivo contenente siero fetale di vitello di 10% (vol/vol), 50 UI/mL penicillina, 50 µ g/mL di miscela di streptomicina) per una concentrazione finale di 1 x 106 cellule/mL.</li>
	<li>Aliquotare ~ 2 x 106 cellule per pozzetto di una piastra ben 6.</li>
	<li>Per indurre l'apoptosi, rimuovere il coperchio della piastra e irradiare le cellule a 150 mJ/cm2 utilizzando un irradiatore UV. Questo dovrebbe prendere circa 30-60 s. Nota: Prima di irradiamento, assicurarsi che ~0.5 x 106 celle vengono mantenute per il controllo di cella 'Non trattato'. Nota: L'apoptosi può essere indotta anche tramite altri metodi quali anti-Fas o siero fame6.</li>
	<li>Incubare a 37° C, 5% CO2 per 2-8 ore, a seconda della linea cellulare.</li>
	<li>Utilizzando un microscopio chiaro superiore di panca, visualizzare le cellule per confermare la presenza di morfologie apoptotici, come blebbing, formazione di apoptopodia e ApoBD. Nota: ingrandimento 40x è sufficiente</li>
	<li>Utilizzando una pipetta P1000, pipettare e raccogliere campioni di apoptotic.</li>
	<li>Lavare la piastra con PBS 1X e si combinano con campione rimanente per garantire il massimo rendimento.</li>
	<li>Raccogliere ~1/10th per il 'intero Apoptotic campione' (WAS) di controllo.</li>
	<li>Raccogliere il campione 'Non trattato'.</li>
	<li>Centrifugare i campioni non trattato sia WAS a 3.000 x g per 6 min.</li>
	<li>Risospendere in 1 mL di PBS 1X e mettere da parte il ghiaccio.</li>
	<li>Per l'isolamento di ApoBD, continuare a passo 2 o 3 con il restante campione di apoptotic.</li>
</ol>2. ApoBD isolamento tramite FACS
<ol><li>Centrifugare il campione intero a 3.000 x g per 6 min.</li>
	<li>Rimuovere la maggior parte del surnatante senza interrompere il pellet.</li>
	<li>Risospendere in una soluzione colorante contenente 1 mL di buffer di associazione x A5 1, 75 µ l di A5-FITC e 2 µ l di TO-PRO-3 a 1 x 107 cellule.</li>
	<li>Incubare il campione al buio a temperatura ambiente per 10 min.</li>
	<li>Aggiungere 1-2 mL di buffer di associazione x A5 1 e centrifugare il campione a 3.000 x g per 6 min rimuovere macchie in eccesso. Nota: Per le popolazioni delle cellule miste o campioni di tessuto, eseguire un anticorpo che macchia passo utilizzando una combinazione di indicatori specifici del tipo delle cellule in 1 tampone di associazione x A5 e incubare in ghiaccio per 20 minuti (o secondo il protocollo del produttore) prima centrifugazione a 3.000 x g per 6 min.</li>
	<li>Risospendere il pellet di campione in 3 mL di tampone di FACS (1x PBS, 1 tampone di associazione x A5, 10% FSC, 2 mM EDTA) per 1 x 107 cellule.</li>
	<li>Filtrare con un colino di cella 70 µm in un fondo di turno, tubo in polipropilene (citometria a flusso) e mantenere i campioni sul ghiaccio e al buio.</li>
	<li>Accendere la macchina di FACS ed eseguire set standard utilizzando un ugello di 100 µm, eseguire il ritardo di goccia e garantire un flusso stabile.</li>
	<li>Caricare l'esempio e impostare la velocità di acquisizione a ~ 1000 eventi/s.</li>
	<li>Regolare FSC, SSC, APC (TO-PRO-3) e tensioni di FITC (A5) e posizionare gli eventi entro le trame di FACS affinché le popolazioni possono essere chiaramente separati.</li>
	<li>Registrare eventi di 20.000.</li>
	<li>Impostare una strategia di gating come in parte 4.</li>
	<li>Nel layout di sorta, è necessario aggiungere l'ultimo cancello di ApoBD come la popolazione di ordinamento desiderata.</li>
	<li>Iniziare l'acquisizione del campione ed eseguire una sorta di prova attraverso la raccolta di 5.000-10.000 ApoBDs in una nuova provetta contenente ~ 250 µ l di tampone FACS.</li>
	<li>Eseguire un sistema torna a filo e carico ordinato ApoBDs.</li>
	<li>Acquisire e registrare test-ordinamento ApoBDs.</li>
	<li>Verificare che la purezza di ApoBD ~ 99% confrontando vs FSC (asse y) A5 (eventi asse x). Nota: A5 macchiatura può ridurre leggermente quando ri-analisi dei campioni a causa di sbiancamento laser.</li>
	<li>Una volta ottenuta la elevata purezza, caricare il campione originale e continuare l'ordinamento fino a quando il numero desiderato di ApoBDs è stato ottenuto. Nota: Se necessario, l'ordinamento dual può essere eseguita per isolare contemporaneamente ApoCells e ApoBDs. Nota: Durante l'ordinamento per un lungo periodo di tempo, si raccomanda di incubare la provetta di raccolta a 4 ° C.</li>
	<li>Una volta che l'ordinamento è completo, raccogliere una piccola porzione di ApoBDs post-ordinamento, post-ordinamento ApoCells, non trattato e WAS apoptosi di convalidare e confermare post-ordinamento purezza. Nota: Anche se la prova-ordinamento e la purezza di post-ordinamento non dovrebbe differire significativamente, questo si basa sulle impostazioni di flusso e stabilità.</li>
</ol>3. ApoBD isolamento tramite centrifugazione differenziale
<ol><li>Centrifugare il campione di apoptotic rimanenti a 300 x g per 10 min.</li>
	<li>Raccogliere il surnatante, lasciando ~ 500 µ l per evitare di interrompere il pellet cellulare e aggiungere in una nuova provetta conica da 15 mL.</li>
	<li>Rimuovere i rimanenti 500 µ l e risospendere il pellet cellulare in 2 mL di PBS 1X (questo rappresenta la frazione di ApoCell-arricchita.</li>
	<li>Centrifugare il surnatante raccolto per 20 min a 3.000 g.</li>
	<li>Controllare una pallina e rimuovere con cautela il surnatante (il supernatante può contenere piccole vescicole extracellulari tra cui microvescicole ed esosomi).</li>
	<li>Risospendere il pellet in 1 mL di PBS 1X (questo rappresenta la frazione 'arricchita di ApoBD')</li>
	<li>Raccogliere 100 µ l di ogni vitali, WAS, arricchita di ApoCell e ApoBD-arricchita di campioni in un nuovo fondo di turno, tubo di polistirolo (citometria a flusso).</li>
	<li>Aggiungere 100 µ l di macchia contenente 2 buffer obbligatorio x A5, 1: 100 A5-FITC e 1:1,000 TO-PRO-3</li>
	<li>Incubare a temperatura ambiente per 10 min al buio.</li>
	<li>Analizzare i campioni mediante citometria a flusso, utilizzando la strategia di gating come descritto in precedenza per convalidare la riuscita induzione di apoptosi e la purezza della frazione arricchita di ApoBD.</li>
</ol>4. flusso Cytometry Gating strategia
<ol><li>Plot TO-PRO-3 (asse y) contro FSC (asse x) per separare le cellule necrotiche (TO-PRO-3alta) da tutti gli altri eventi non-permeabilized (TO-PRO-3basso/intermedio).</li>
	<li>Selezionare tutti gli eventi non-permeabilized e trama SSC contro A5. Due popolazioni tra cui popolazione 1 (P1), SSCintermedio/alto, A5basso/intermedio celle e popolazione 2 (P2), tutti gli altri eventi del cancello.</li>
	<li>Da P2, plot TO-PRO-3 contro A5 e selezionare A5intermedio/alto eventi per escludere tutti i detriti cellulari.</li>
	<li>Selezionare tutti gli eventi positivi A5 e plot FSC contro A5. Separare ApoBDs (FSCbasso) da ApoCells (FSCintermedio/alto). Nota: Quando gating ApoBDs per l'isolamento di ApoBD tramite l'approccio basato su FACS, consigliamo un passaggio finale selezionando ApoBDs e confronto a-PRO-3-A5 e tutti gli eventi. Questo assicura che il cancello di ordinamento finale utilizza fluorescenza piuttosto che i parametri FSC/SSC.</li>
	<li>Per l'analisi di cellule vitali, selezionare P1 ed effettuare una delle due strategie di Gate. Per l'analisi generale delle cellule vitali, plot FSC contro A5 e selezionare tutte le celle FSCintermedio/alto , quindi rimozione detriti cellulari rimanenti. In alternativa, per un'analisi approfondita, cellule vitali possono essere separate da A5– ApoCells primi tracciando TO-PRO-3 contro FSC. Selezionare a-PRO-3basso, cellule vitali di FSCintermedio/alto e TO-PRO-3intermedio, FSCintermedio/alto A5– ApoCells presto.</li>
</ol>

<ol>
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Dalla sua descrizione iniziale nel 1950, il campo dell'apoptosi ha avanzato notevolmente, diventando una zona prominenti di ricerca. Nonostante il vasto interesse e gli sforzi profusi, alcuni aspetti dell'apoptosi, in particolare la formazione di ApoBDs, non sono stati studiati bene a causa della mancanza di adeguate metodologie. Queste comprendono in particolare la limitazione in progressione di apoptosi e ApoBD formazione contemporaneamente usando la citometria a flusso tradizionale analisi A5/PI e le impurità di isol...

Apoptosi, una forma ben studiata di morte cellulare programmata, sono necessaria per mantenere l'omeostasi fisiologica e rimuovere le cellule potenzialmente dannose all'interno del corpo umano1. Dopo l'induzione dell'apoptosi, cellule apoptotiche (ApoCells) possono subire una serie di cambiamenti morfologici e smontare in piccole vescicole di membrana-limita, chiamate ApoBDs. Nel complesso, questo processo è noto come smontaggio delle cellule apoptotiche e può essere diviso in 3 fasi distinte sulla base di morfologia2,3. Passaggio 1 (membrana plasmatica blebbing) è caratterizzata dalla formazione di aerostato-come le strutture sulla superficie delle cellule, noto come vescichette4,5. Passaggio 2 (formazione di protrusione apoptotici) comprende la formazione di protrusioni di membrana lungo come apoptopodia, apoptopodia in rilievo e microtubuli picchi6,7,8. Infine, passo 3 (formazione di ApoBD) include la frammentazione apoptotica protrusioni e/o ApoCells per generare ApoBDs6,9. I risultati precedenti hanno suggerito un ruolo di ApoBDs in favoreggiamento liquidazione delle cellule apoptotiche e mediare la comunicazione intercellulare. Ad esempio, si propone che la frammentazione di un ApoCell in ApoBDs può generare piccoli pezzi 'bite-sized' che potrebbero essere facilmente rimossi da circostante fagociti2,10,11. Inoltre, ApoBDs può harbor una serie di biomolecole quali DNA, RNA e proteine, che possono essere vittime di tratta a cellule circostanti per facilitare cella comunicazione12,13,14. Per funzionalmente studiare questi processi, è fondamentale per confermare tre parametri chiave, tra cui (i) validazione di induzione di apoptosi e ApoBD formazione, (ii) isolamento di ApoBDs e (iii) la conferma dell'ApoBD purezza.
In precedenza, una serie di metodi tra cui citofluorimetria e microscopia elettronica è stata usata per studiare l'apoptosi e ApoBDs15,16,17,18. Tuttavia, quantificazione e rilevamento di ApoBD sono spesso difficili o trascurato. Per esempio, l'apoptosi di base di citometria a flusso più ordinariamente utilizzati test impiega Annessina V (A5, una proteina che lega l'esternalizzati ' mangiare-me' segnale fosfatidilserina (PtdSer)) e dell'acido nucleico macchia propidio ioduro (PI)19. Tuttavia, con questa combinazione di macchia universale, analisi presuppone che ci sono solo tre tipi di sottoinsiemi di cellule (cellule vitali, ApoCells e cellule necrotiche) nel campione. Inoltre, anche se considerata come "gold standard" da molti ricercatori per apoptosi, analisi di citometria a flusso e l'analisi successiva dei dati spesso esclude ApoBDs attraverso un passaggio di gating iniziale selezionando eventiintermedio-alto FSC/SSC. Di conseguenza, abbiamo recentemente sviluppato un'analisi di citometria a flusso romanzo usando A5 e TO-PRO-3, un'altra macchia di acido nucleico che può essere preso in modo selettivo di caspase 3/7-attivato Pannexina 1 (PANX1) canali7,20. Come attivazione di caspase 3-induced PANX1 precede l'esposizione PtdSer nella fase iniziale dell'apoptosi, TO-PRO-3 macchie differenzialmente apoptotici e cellule necrotiche. Inoltre, questo approccio combinato con il nostro romanzo gating strategia include eventi tutti acquisiti durante l'analisi dei dati e di conseguenza, sei sottoinsiemi di cellule/particella sono identificati, tra cui: (i) cellule vitali (FSC/SSCintermedio/alto, A5basso , A-PRO-3bassa), (ii) A5– primi ApoCells (FSC/SSCintermedio/alto, A5basso, TO-PRO-3intermedi), (iii) A5+ ApoCells (FSC/SSCintermedio/alto, A5alta , A-PRO-3intermedi), cellule necrotiche (iv) o tardo ApoCells (FSC/SSCintermedio/alto, A5alta, TO-PRO-3alta), (v) ApoBDs (FSC/SSCbasso, A5intermedi, a-PRO-3basso / intermedio) e (vi) detriti (FSC/SSCbasso, A5basso, TO-PRO-3basso)20. Il nostro approccio sottolinea l'importanza di analizzare tutti i sottoinsiemi di cellule/particella e, cosa ancora più importante, la separazione di ApoBDs dalle cellule e detriti20. Quindi, questo approccio dimostra una tecnica efficace per convalidare l'induzione di apoptosi e ApoBD formazione simultaneamente.
Tradizionalmente, i ApoBDs sono stati isolati attraverso una varietà di approcci di centrifugazione differenziale per cui ApoBDs può essere separato dalle cellule o altre vescicole extracellulari in base alla densità. Tuttavia, tali metodi di centrifugazione sono spesso limitati dalla bassa ApoBD purezza, mancanza di una fase di quantificazione per confermare la purezza del campione e/o incapacità di separare cella tipo-specific ApoBDs17,21,22. Di conseguenza, abbiamo sviluppato recentemente due approcci, basati su FACS e un nuovo approccio differenziale basato su centrifugazione che può essere accoppiato con il nostro metodo di citometria a flusso precedentemente stabilito per convalidare l'induzione di apoptosi e campione purezza23. ApoBD isolamento tramite nostro approccio basato su FACS può arricchire ApoBDs fino a 99% di purezza e può essere accoppiato con una varietà di anticorpi specifici del tipo delle cellule per isolare ApoBDs da popolazioni di cellule miste, campioni di tessuti e fluidi corporei23. Inoltre, il nostro approccio riveduto centrifugazione differenziale viene illustrato un metodo efficiente per isolare ApoBDs a > 90% purezza23.
In questo articolo, descriviamo dettagliatamente la nostra procedura sperimentale per convalidare l'induzione di apoptosi e per rilevare e quantificare ApoBD formazione. I flussi di lavoro di isolamento ApoBD utilizzando metodi basati su centrifugazione differenziali e basati su FACS anche elaborati e confrontati. I dati rappresentativi dimostrano che la metodologia descritta fornisce un efficace strumento all'avanguardia per gli studi futuri di ApoBD.

<table><tbody><tr><td>Cells e.g. cultured human THP-1 monocytes (clone number: TIB-202)</td><td>ATCC</td><td>-</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Life Technologies</td><td>22400-089</td><td></td></tr><tr><td>Penicillin-streptomycin mixture</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>FSC</td><td>Gibco</td><td>10099-141</td><td></td></tr><tr><td>1x PBS</td><td>-</td><td>-</td><td></td></tr><tr><td>Annexin V FITC</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>TO-PRO-3 iodide</td><td>Life Technologies</td><td>T3605</td><td>TO-PRO-3 may cause skin, eye and respiratory irration. Avoid direct contact.</td></tr><tr><td>10x Annexin V binding buffer</td><td>BD Bioscience</td><td>556454</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>1001710526</td><td></td></tr><tr><td>Centrifuge tube (15 mL)</td><td>Cellstar</td><td>188271</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 mL)</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>Tissue culture incubator (37 &amp;deg;C, 5% CO2)</td><td>-</td><td>-</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>392932</td><td></td></tr><tr><td>FACS ARIA III Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Canto II Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Diva 6.1.1 software</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FlowJo 8.8.6 software</td><td>-</td><td>-</td><td></td></tr><tr><td>&amp;nbsp;UV Stratalinker 1800</td><td>Stratagene</td><td>-</td><td></td></tr></tbody></table>

detection and isolation of apoptotic bodies to high purity

I membri principali della famiglia delle vescicole extracellulari, con ApoBDs essendo uno del più grande tipo di corpi apoptotici (ApoBDs), microvescicole ed esosomi. È stato proposto che ApoBDs può aiutare la liquidazione delle cellule, come pure la comunicazione intercellulare attraverso traffico biomolecole. Gli approcci convenzionali utilizzati per l'identificazione e l'isolamento di ApoBDs sono spesso limitati dalla mancanza di quantificazione accurata e purezza basso del campione. Qui, descriviamo un flusso di lavoro per confermare l'induzione di apoptosi, convalidare ApoBD formazione e isolare ApoBDs di elevata purezza. Inoltre, contorni e confrontare approcci di centrifugazione differenziale basato per isolare ApoBDs e fluorescenza-attivato delle cellule ordinano (FACS). Inoltre, la purezza del ApoBDs isolato verrà confermata utilizzando un stabilire in precedenza flusso cytometry-based colorazione e metodo analitico. Presi insieme, utilizzando l'approccio descritto, THP-1 del monocito apoptosi e apoptotica delle cellule lo smontaggio è stato indotto e validato, e ApoBD generati dai monociti THP-1 sono stati isolati per una purezza del 97-99%.

Utilizzando la procedura descritta qui, è stato indotto apoptosi monociti THP-1 e ApoBDs sono stati rilevati e isolato tramite un FACS-based o un approccio di centrifugazione differenziale (Figura 1). In primo luogo, l'apoptosi è stata indotta da irradiazione UV e campioni sono stati raccolti dopo 2-3 h di incubazione, quando le cellule hanno esibito apoptotica morfologie, compreso blebbing, formazione di protrusione della membrana apoptotici e la generazio...

Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

Detection and Isolation of Apoptotic Bodies to High Purity

Un flusso di lavoro utilizzando una centrifuga di flusso cytometry o differenziale è stato sviluppato per individuare, quantificare e isolare i corpi apoptotici da un campione di apoptotic di elevata purezza.

Rilevamento e isolamento di corpi apoptotici di elevata purezza

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

detection-and-isolation-of-apoptotic-bodies-to-high-purity

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

10.5K Views.  La Trobe University. Un flusso di lavoro utilizzando una centrifuga di flusso cytometry o differenziale è stato sviluppato per individuare, quantificare e isolare i corpi apoptotici da un campione di apoptotic di elevata purezza.

Video: Detection and Isolation of Apoptotic Bodies to High Purity

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Flusso Cytometric Detection neonata al seno cancro cellule staminali-come delle cellule dopo l'inversione di apoptosi

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Ricerca

Ricerca sul cancro

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

Monitoraggio spaccati Caspase-3 Attività e apoptosi di cellule oligodendrogliali Immortalata dal vivo utilizzando cellule Imaging e Cleaveable fluorogenici-dye seguenti supporti di potassio indotta depolarizzazione della membrana

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Neuroscienze

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Analisi citometrica a flusso di biomarcatori apoptotici in cellule di cancro cervicale SiHa trattate con attinomicina D

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

L&#39;uso di una caspasi Multiplexing test per determinare apoptosi in un modello cellulare ipotalamica

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

Misurazione dell'attività della caspasi mediante un saggio fluorometrico o citometria a flusso

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Biochimica

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

Modificato annessina V / propidio ioduro Apoptosis analisi di valutazione accurata di morte cellulare

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Biologia

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantificazione del fagosoma da microscopia di fluorescenza di cella singola

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Immunologia e infezioni

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Analisi sperimentale di apoptotic thymocyte engulfment da macrofagi

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Strategie per l&#39;inseguimento Anastasis, una cellula di sopravvivenza fenomeno che Inverte Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Identificazione di segnale intracellulare eventi indotta in cellule vitali per interazione con le cellule vicine sottoposti apoptotica delle cellule morte

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Rilevamento e isolamento di corpi apoptotici di elevata purezza

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