Gli autori ringraziano Jose Luis De la Vega, Erika Melchy e Dr. Takuya Nishigaki per l&#39;assistenza tecnica. Questo lavoro è stato supportato dal Consejo Nacional de Ciencia y Tecnología (CONACYT-Messico) (99.333 e 128.566 per CT); Dirección General de Asuntos del personale Académico / Universidad Nacional Autónoma de México (IN202212-3 a CT).

<ol>
	<li>Bouschet, T., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+as+an+extracellular+signalling+molecule:+perspectives+on+the+Calcium+Sensing+Receptor+in+the+brain.">Calcium as an extracellular signalling molecule: perspectives on the Calcium Sensing Receptor in the brain.</a> Comptes Rendus Biologies. 328, 691-700 (2005).</li><li>Darszon, A., Nishigaki, T., Beltran, C., Trevino, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+channels+in+the+development,+maturation,+and+function+of+spermatozoa.">Calcium channels in the development, maturation, and function of spermatozoa.</a> Physiol. Rev. 91, 1305-1355 (2011).</li><li>Esposito, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+deficient+for+soluble+adenylyl+cyclase+are+infertile+because+of+a+severe+sperm-motility+defect.">Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 2993-2998 (2004).</li><li>Avenarius, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+male+infertility+caused+by+mutations+in+the+CATSPER1+channel+protein.">Human male infertility caused by mutations in the CATSPER1 channel protein.</a> American Journal of Human Genetics. 84, 505-510 (2009).</li><li>Carlson, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+targeting+of+native+CatSper+channels+reveals+a+required+role+in+maintenance+of+sperm+hyperactivation.">Pharmacological targeting of native CatSper channels reveals a required role in maintenance of sperm hyperactivation.</a> PLoS ONE. 4, e6844 (2009).</li><li>Brokaw, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+flagellar+response+during+the+chemotaxis+of+bracken+spermatozoids.">Calcium and flagellar response during the chemotaxis of bracken spermatozoids.</a> J. Cell. Physiol. 83, 151-158 (1974).</li><li>Visconti, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacitation+of+mouse+spermatozoa.+II.+Protein+tyrosine+phosphorylation+and+capacitation+are+regulated+by+a+cAMP-dependent+pathway.">Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.</a> Development. 121, 1139-1150 (1995).</li><li>Svahn, H. A., van den Berg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cells+or+large+populations.">Single cells or large populations.</a> Lab on a chip. 7, 544-546 (2007).</li><li>Pepperkok, R., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+fluorescence+microscopy+for+systems+biology.">High-throughput fluorescence microscopy for systems biology.</a> Nat. Rev. Mol. Cell Biol. 7, 690-696 (2006).</li><li>Strunker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CatSper+channel+mediates+progesterone-induced+Ca2++influx+in+human+sperm.">The CatSper channel mediates progesterone-induced Ca2+ influx in human sperm.</a> Nature. 471, 382-386 (2011).</li><li>Lishko, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Control+of+Male+Fertility+by+Spermatozoan+Ion+Channels.">The Control of Male Fertility by Spermatozoan Ion Channels.</a> Annu. Rev. Physiol. , (2011).</li><li>Kao, J. P., Harootunian, A. T., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+generated+cytosolic+calcium+pulses+and+their+detection+by+fluo-3.">Photochemically generated cytosolic calcium pulses and their detection by fluo-3.</a> J. Biol. Chem. 264, 8179-8184 (1989).</li><li>Kilic, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caged+progesterone:+a+new+tool+for+studying+rapid+nongenomic+actions+of+progesterone.">Caged progesterone: a new tool for studying rapid nongenomic actions of progesterone.</a> Journal of the American Chemical Society. 131, 4027-4030 (2009).</li><li>Lishko, P. V., Botchkina, I. L., Kirichok, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+activates+the+principal+Ca2++channel+of+human+sperm.">Progesterone activates the principal Ca2+ channel of human sperm.</a> Nature. 471, 387-391 (2011).</li><li>Xia, J., Ren, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BSA-induced+Ca2++influx+during+sperm+capacitation+is+CATSPER+channel-dependent.">The BSA-induced Ca2+ influx during sperm capacitation is CATSPER channel-dependent.</a> Reprod. Biol. Endocrinol. 7, 119 (2009).</li><li>Galantino-Homer, H. L., Florman, H. M., Storey, B. T., Dobrinski, I., Kopf, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+sperm+capacitation:+assessment+of+phosphodiesterase+activity+and+intracellular+alkalinization+on+capacitation-associated+protein+tyrosine+phosphorylation.">Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation.</a> Mol. Reprod. Dev. 67, 487-500 (2004).</li><li>Baldi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+accumulation+and+responsiveness+to+progesterone+in+capacitating+human+spermatozoa.">Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa.</a> J. Androl. 12, 323-330 (1991).</li></ol>

Non abbiamo nulla da rivelare.

Segnalazione intracellulare è di vitale importanza per la maggior parte delle attività cellulari; Ca 2 + è un messaggero ubiquitario che accompagna le cellule di mammiferi in tutto il loro intero ciclo di vita, dalla loro origine alla fecondazione, alla fine del loro ciclo di vita. In risposta a stimoli diversi, [Ca 2 +] i aumenta, oscilla e diminuisce con codificazione spazio-temporale, di conseguenza, diversi processi sono attivati, modulate o denuncia da parte di Ca 2 + messaggi co...

Ca 2 + è un secondo messaggero universale di vie di trasduzione del segnale in cellule eucariotiche. Intracellulare di Ca 2 + (Ca 2 + i) partecipa alla regolazione di molti processi fisiologici fondamentali nelle cellule sia eccitabili e non eccitabili. L&#39;importanza e l&#39;universalità di Ca 2 + come secondo messaggero durante eventi di trasduzione del segnale è derivato dalla sua versatilità spazio-temporale nella trasmissione delle informazioni all&#39;interno della cellula. Mentre Ca 2 + non possono essere sintetizzati de novo o degradati all&#39;interno della cellula, la sua concentrazione intracellulare ([Ca 2 +] i) viene mantenuta entro limiti molto ristretti attraverso diversi meccanismi cellulari che continuamente tampone, sequester, compartimenti stagni, e / o accumulare Ca 2 +. Cambiamenti nella concentrazione di questo ione possono verificarsi in aree molto localizzate all&#39;interno della cella 1, e decifrare tali fluttuazioni è essenziale per ottenere una deeper comprensione di (1) il loro ruolo nel meccanismo di segnalazione, (2) il loro significato fisiologico, e (3) meccanismi generali di segnalazione cellulare. Ca 2 +-mediata segnalazione è di particolare importanza in sperma fisiologia 2. La motilità degli spermatozoi è una delle funzioni più importanti per il successo la fecondazione, e in effetti, diversi difetti della motilità degli spermatozoi può causare sterilità 3-5. L&#39;importanza di Ca 2 + in movimento flagellare tempo è stato riconosciuto 6, tuttavia, il meccanismo di come Ca 2 + controlla la forma specifica di flagellare curvatura non è completamente nota. Prima di fondersi con l&#39;uovo, spermatozoi deve subire capacitazione, un processo complesso alla residenza spermatozoo all&#39;interno del tratto femminile. Durante la capacitazione, architettura lipidico della membrana e l&#39;organizzazione spermatozoi vengono modificati, soprattutto a seguito della rimozione del colesterolo dalla membrana plasmatica. Inoltre, diverse proteine ​​sono tirosin-fosforoylated 7. Soprattutto, durante la capacitazione vi è un aumento del pH intracellulare (pH i) e in [Ca 2 +] i, e il potenziale di membrana hyperpolarizes in alcune specie 2. Capacitazione avviene solo in una sottopopolazione di spermatozoi (20-40%), ed i meccanismi coinvolti in tutti questi cambiamenti cellulari sono tutt&#39;altro che chiare. E &#39;generalmente accettato che solo una sottopopolazione di spermatozoi capacitati subiscono la reazione acrosomiale (AR), quando esposti ad induttori fisiologici. L&#39;AR è anche un evento di 2 +-regolamentato Ca richiesto per la fertilizzazione in tutte le specie che possiedono un acrosomiale (organello specializzato con le membrane esterne ed interne). Durante questo processo i fusibili membrana acrosomiale esterna con la membrana plasmatica dello spermatozoo, rilasciando enzimi idrolitici che permettono al spermatozoo di penetrare la matrice glico-proteico che circonda l&#39;uovo (zona pellucida, o ZP). L&#39;AR espone anche una nuova superficie spermatozoo fusogenica che interagisce conla membrana plasmatica uovo per la fusione finale di entrambi i gameti. Ci sono diversi ligandi cellulari che inducono l&#39;AR, progesterone essendo uno dei più studiati tra loro. In questo lavoro presentiamo quattro diverse tecniche che prevedono l&#39;utilizzo di un colorante fluorescente Ca 2 +-sensibile da misurare [Ca 2 +] i cambiamenti di sperma umano innescata dal progesterone (tranne per citometria di flusso, in cui abbiamo misurato la [Ca 2 + ] i aumentare indotta durante il processo di capacitazione in vitro). In questo caso particolare abbiamo utilizzato Fluo-3 AM (Life Technologies, Grand Island, NY), un colorante membrana permeabile con una K d = 325 nM. In vitro abbiamo monitorato cambiamenti di fluorescenza in funzione del tempo con tre delle metodologie, e con la quarta tecnica abbiamo misurato valori di fluorescenza in un singolo punto nel tempo dato. Questi diversi approcci sono complementari, in quanto del tutto che forniscono risoluzione spaziale e temporaleoluzione sia a singola cella e livelli di popolazione di cellule. Cella Popolazione o esperimenti Bulk Tecniche di bulk sono ampiamente utilizzati non solo perché gli strumenti di cui hanno bisogno sono facilmente disponibili, ma anche perché sono semplici, ben consolidata, e consentono il calcolo della media delle informazioni a misurazioni effettuate su milioni di cellule in un singolo esperimento. Tecnica # 1. Fluorometria convenzionale Questa tecnica monitora le variazioni nella fluorescenza in funzione del tempo; gli esperimenti sono eseguite in provette di vetro con volumi di campione da 200 a 1.000 microlitri. Corretta miscelazione dei reagenti aggiunti richiede agitazione magnetica, e quindi la risoluzione temporale ottenuta è dell&#39;ordine di secondi. L&#39;intervallo di concentrazione cellulare tipica dei campioni analizzati è di 10 -10 8 5 cellule / ml. Tecnica # 2. Arrestato fluorometria flusso Tsua tecnica controlla anche cambiamenti nella fluorescenza in funzione del tempo, ma i reagenti vengono rapidamente miscelati tra loro (mediante pressione) in una cuvetta di registrazione contenente un volume di campione molto piccolo (che vanno 25-100 microlitri). Pertanto, omogeneizzazione dei reagenti è istantanea, consentendo un&#39;elevata risoluzione temporale nell&#39;ordine di millisecondi. Analisi delle tracce risultanti fluorescenza in funzione del tempo sono adatte per determinare le velocità di reazione, chiarire la complessità del meccanismo di reazione, ottenendo informazioni sulle intermedi di reazione breve durata, ecc L&#39;intervallo di concentrazione cellulare comune dei campioni analizzati è 10 5 -10 7 cellule / ml. Esperimenti Single Cell Esperimenti bulk riportano il comportamento medio di un gran numero di cellule, tuttavia, una popolazione può frequentemente esibire proprietà eterogenee che sono trascurati durante tale tipo di misure. Tecniche di singola cella sono quindi utilizzati per integrare thinformazioni e ottenuto con esperimenti popolazioni cellulari. Tecnica # 3. Citometria a Flusso Nonostante l&#39;importanza delle informazioni derivanti da misurazioni singola cellula, è importante analizzare un gran numero di celle per impedire l&#39;estrapolazione erronea proprietà cellula-specifici per un&#39;intera popolazione. Per questo motivo, le tecniche ad alta produttività sono favorite e il metodo più popolare è citometria di flusso, in cui 10.000 cellule per condizione sono convenzionalmente analizzati. Questo metodo consente l&#39;analisi multi-parametrica di popolazioni eterogenee come è categorizza le cellule in base alla loro dimensione (forward scatter (FSC)), granularità (side scatter (SSC)) e di intensità di fluorescenza (etichettatura specifica con un anticorpo, marcatore viabilità, ecc) , fornendo così informazioni sulla distribuzione dei parametri &#39;per un gruppo di celle. Citometria a flusso fornisce immediata, piuttosto che le informazioni in funzione del tempo 8. In avanti e laterale valori scatter are anche utile per selezionare una porta che include celle ma discrimina detriti cellulari, polvere, ecc Per misure di fluorescenza, fluorescenza controlli negativi e positivi deve essere inclusa anche. Se viene usato più di un canale di fluorescenza, un processo noto come compensazione deve essere eseguita (per dettagli vedere <a href="http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp" target="_blank">http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp</a> ). Compensazione consente sovrapposizione spettrale discriminazione tra fluorofori. Citometria di flusso consente anche la discriminazione di cellule morte, generalmente per mezzo di propidio ioduro colorazione. Tecnica # 4. Singolo Imaging cellulare Microscopia è un altro metodo comune per studiare il comportamento di singole cellule, ma è adatto per gli studi di tempo-dipendenti e fornisce anche la risoluzione spaziale. Un grave inconveniente è che l&#39;analisi high-throughput è solo agli inizi in questo momento <sup&gt; 9.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Ham's F-10</td>
 <td>Sigma-Aldrich</td>
 <td>N-6013</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A-7906</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride Dihydrate approx. 99%</td>
 <td>Sigma-Aldrich</td>
 <td>C-3881</td>
 <td></td>
 </tr>
 <tr>
 <td>Makler Counting Chamber</td>
 <td>SEFI Medical Insruments LTD</td>
 <td>SEF-MAKL</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-3 AM</td>
 <td>Invitrogen</td>
 <td>F-1242</td>
 <td>20 vials/50 &mu;g each</td>
 </tr>
 <tr>
 <td>Ionomycin</td>
 <td>Alomone</td>
 <td>I-700</td>
 <td></td>
 </tr>
 <tr>
 <td>Progesterone</td>
 <td>Sigma-Aldrich</td>
 <td>P0130</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>S-9888</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Potassium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>P-3911</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>JT Baker</td>
 <td>3506</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Magnesium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>M-2670</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Calcium chloride anhydrous</td>
 <td>Sigma-Aldrich</td>
 <td>C-1016</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H-3125</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>D-Glucose</td>
 <td>JT Baker</td>
 <td>1906-01</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium pyruvate</td>
 <td>Sigma-Aldrich</td>
 <td>P-2256</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium L-lactate (aprox. 99%)</td>
 <td>Sigma-Aldrich</td>
 <td>L- 7022</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Propidium Iodide </td>
 <td>Invitrogen</td>
 <td>L-7011 </td>
 <td>Component B</td>
 </tr>
 <tr>
 <td>Triton X-100 (t-Octylphenoxypolyethoxyethanol)</td>
 <td>Sigma- Aldrich</td>
 <td>X-100</td>
 <td>2.4 mM solution in water </td>
 </tr>
 <tr>
 <td>Round coverslip </td>
 <td>VWR</td>
 <td>48380 080</td>
 <td>25 mm diameter</td>
 </tr>
 <tr>
 <td>Poly-L-lysine solution </td>
 <td>Sigma-Aldrich</td>
 <td>P8920</td>
 <td></td>
 </tr>
 <tr>
 <td>Manganese chloride</td>
 <td>Sigma-Aldrich</td>
 <td>M-3634</td>
 <td></td>
 </tr>
 <tr>
 <td>Attofluor; Cell Chamber, for microscopy</td>
 <td>Life technologies</td>
 <td>A-7816</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl Sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>5x5 ml</td>
 </tr>
 </tbody>
</table>

measuring intracellular ca2+ changes in human sperm using four techniques: conventional fluorometry, stopped flow fluorometry, flow cytometry and single cell imaging

Gli spermatozoi sono cellule riproduttive maschili specialmente progettati per raggiungere, riconoscere e fondersi con l&#39;uovo. Per eseguire queste attività, gli spermatozoi devono essere preparati ad affrontare un ambiente in costante evoluzione e per superare le numerose barriere fisiche. Essere in sostanza transcriptionally e translationally silenziosa, queste cellule mobili affidano profondamente su diversi meccanismi di segnalazione di orientarsi e di nuotare in modo diretto, e fare i conti con le difficili condizioni ambientali durante il loro viaggio per trovare l&#39;uovo. In particolare, Ca 2 +-mediata segnalazione è fondamentale per diverse funzioni spermatiche: attivazione della motilità, capacitazione (un processo complesso che prepara lo sperma per la reazione acrosomiale) e la reazione acrosomiale (un evento esocitotico che permette la fusione spermatozoo-uovo). L&#39;uso di coloranti fluorescenti per monitorare fluttuazioni intracellulari di questo ione è di notevole importanza per la loro facilità di applicazione, sensibilità e versatilità di detezione. Utilizzando un unico protocollo di colorante di caricamento utilizziamo quattro diverse tecniche di fluorimetrici per monitorare lo sperma Ca 2 + dinamica. Ciascuna tecnica fornisce informazioni distinta che consente la risoluzione spaziale e / o temporale, generando dati sia a singola cella e di popolazioni cellulari.

<img alt="Figura 1" fo:content-width="6in" fo:src="/files/ftp_upload/50344/50344fig1highres.jpg" src="/files/ftp_upload/50344/50344fig1.jpg" /> Figura 1. Rappresentazione schematica del protocollo sperimentale per la preparazione dei campioni di sperma con il metodo semi-sommerso. Le fasi principali per la separazione di spermatozoi mobili e per la regolazione della loro concentrazione sono illustrate. L&#39;ultimo passaggio di incubazione viene eseguita solo q...

Watch this Scientific Journal Video about Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell 

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellulare di Ca<sup&gt; 2 +</sup&gt; Dinamiche sono molto importanti in sperma fisiologia e Ca<sup&gt; 2 +</sup&gt; Sensibili coloranti fluorescenti costituiscono uno strumento versatile per studiarli. Esperimenti di popolazione (fluorometria e fermato il flusso fluorometria) ed esperimenti cella singola (citometria a flusso e l&#39;imaging di singola cellula) sono utilizzati per tenere traccia spazio-temporale [Ca<sup&gt; 2 +</sup&gt;] Cambiamenti nelle cellule di sperma umano.

Misurare intracellulare Ca<sup&gt; 2 +</sup&gt; Modifiche di sperma umano utilizzando quattro tecniche: fluorometria convenzionale, si fermò fluorometria Flow, Citometria a flusso singolo e l&#39;imaging cellulare

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

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Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

20.1K Views.  Instituto de Biotecnología-Universidad Nacional Autónoma de México. Intracellulare di Ca 2 + Dinamiche sono molto importanti in sperma fisiologia e Ca 2 + Sensibili coloranti fluorescenti costituiscono uno strumento versatile per studiarli. Esperimenti di popolazione (fluorometria e fermato il flusso fluorometria) ed esperimenti cella singola (citometria a flusso e l'imaging di singola cellula) sono utilizzati per tenere traccia spazio-temporale [Ca 2 +] Cambiamenti nelle cellule di s...

Video: Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Tecniche di Imaging Ca 2 + Segnalazione di sperma umano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Ricerca

Biologia

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purificazione Citometria a flusso di cellule di topo Meiotic

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Cella di misura Kinetics progressione del ciclo con la marcatura metabolica e citometria a flusso

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Ottimizzato Metodi di modellazione e colorazione proliferazione per il monitoraggio divisione cellulare utilizzando coloranti Tracking Cellulari

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

Il<em&gt; Xenopus</em&gt; Ovociti Cut-open vaselina Gap Tecnica Voltage-clamp Con fluorometria

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Tecniche per l&#39;Analisi dei extracellulare vescicole citometria a flusso

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Valutare il flusso autofagico misurando LC3, p62 e LAMP1 Co-localizzazione usando la citometria del flusso di imaging multispettrale

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplicazione tramite flusso Cytometry di tubero isolato protoplasti di misura

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Applicazioni di mappatura spazio-temporale e tecniche di analisi delle particelle per quantificare la segnalazione intracellulare Ca2 + In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...