We thank Dr. Gilles Dupuis for critical reading of the manuscript. 
Funding
This work was supported by the Canadian Institutes for Health Research, grant # MOP-126173. C.M.D is a member of the Fonds de la Recherche en Sant&#233; (FRSQ)-funded Centre de Recherche Clinique &#201;tienne LeBel.

1. Préparation des solutions pour cellulaire pH Calibration <ol><li> Ajouter les composés suivants à cinq tubes de 50 ml coniques distincts 5 pour préparer des solutions à utiliser pour l&#39;étalonnage: <ul><li> De NaCl (1 ml de NaCl 1 M) (1 M NaCl = 0,58 g dans 10 ml H 2 0) </li><li> KCl (6,75 ml de KCl 1 M) (1 M KCl = 0,75 g dans 10 ml H 2 0) </li><li> Glucose (1 ml de 1 M de D-glucose) (1M D-Glucose = 1,80 g dans 10 ml H 2 0) </li><li> MgS0 4 (0,05 ml de 1 M de MgSO 4) (1 M MgSO 4 = 1,20 g dans 10 ml H 2 0) </li><li> HEPES 20 mM (1 ml d&#39;HEPES 1M) (1 M HEPES = 2,38 g dans 10 ml H 2 0) </li><li> CaCl 2 (0,05 ml de CaCl2 1 M) (1 M CaCl2 = 1,47 g dans 10 ml H 2 0) Préparer toutes les solutions double distillée H 2 O (le trou DDH 2 O). </li></ul></li><li> Remplissez chaque solution avec 35 ml de ddH 2 O et mélanger avec un agitateur magnétique jusqu&#39;à sortelution est atteint. </li><li> Ajuster le pH de chaque solution de KOH 1 N ou HCl 1 N pour obtenir des valeurs de pH de 5,5, 6,0, 6,5, 7,0 et 7,5. Ensuite, régler le volume avec de l&#39;eau déminéralisée à un volume final de 50 ml. </li><li> Mesure 5 mg de nigéricine et l&#39;ajouter à 670 pi d&#39;EtOH absolu pour préparer un 10 mM nigéricine solution stock. Attention: nigéricine est très toxique et doit être manipulé avec grand soin (gants, lunettes de protection et masque). Mélanger la solution par inversion jusqu&#39;à la nigéricine est complètement dissous. </li><li> Ajouter 0,05 ml de 10 mM de nigéricine (concentration finale de 10 pM) à chaque solution d&#39;étalonnage. Nigéricine est un ionophore qui augmente la perméabilité de la membrane cellulaire et provoque l&#39;équilibration du pH intracellulaire et extracellulaire en présence d&#39;une concentration de dépolarisation de K + extracellulaire. </li><li> Les solutions d&#39;étalonnage peuvent être stockés jusqu&#39;à 1 mois à 4 ° C </li></ol> 2. Préparation des cellules <ol><li> En vertu d&#39;un Safet biologiquey capot, des cellules de semences en 35 mm x boîtes de culture de 10 mm dans 2 ml de milieu de croissance supplémenté par 10% de sérum et d&#39;antibiotiques de veau fœtal. Utiliser la quantité de cellules nécessaire pour assurer environ 50% de confluence après 24 h. NOTE: Dans nos expériences, nous utilisons la lignée cellulaire HT-1080 et la plaque 1 x 10 5 cellules par boîte de culture. </li><li> Déterminer le nombre de boîtes de culture nécessaires pour l&#39;expérience et la plaque 5 boîtes de culture supplémentaires qui seront utilisés pour l&#39;étalonnage. </li><li> Plats place de la culture dans un C / 5% de CO 2 incubateur à 37 ° pendant au moins 24 heures. </li></ol> 3. Préparation des sondes de détection de pH <ol><li> Préparer une solution stock de 1M de la sonde de pH fluorescent 8 hydroxypyrène-1, 3,6 trisulfonique, sel trisodique (HHP / pyranine) dans le trou DDH 2 0. Cette solution doit être conservé à température ambiante et protégé de la lumière. </li><li> Préparer une solution stock de 1 mM 5 - (et 6)-carboxy seminaphthorhodafluor acétoxyméthylique ester acétate (C-SNARF-une heures; appelé Thereafter SNARF-1) en dissolvant 1 mg de SNARF-1 dans 1,76 ml de DMSO. Bien mélanger par pipetage répété. SNARF-1 solution doit être conservé à -20 ° C et protégé de la lumière. </li></ol> 4. Portable étiquetage <ol><li> Seize heures avant imagerie des cellules vivantes, ajouter 2 pi de 1M HPTS solution stock (concentration finale 1 mM) à chaque boîte de culture contenant 2 ml de milieu complet. </li><li> Retour des boîtes de culture à l&#39;incubateur. La sonde est HPTS cellulaire imperméant, donc étiquette organites la sonde doit être prise par pinocytose. Cette condition exige que les incubations de cellules avec HPTS sont effectuées dans un milieu de culture additionné de sérum. </li><li> Après incubation avec HPTS, laver les cellules deux fois avec du PBS stérile et ajouter 2 ml de milieu exempt de sérum pendant au moins 2 heures. L&#39;incubation avec SNARF-1 doit être effectuée dans un milieu sans sérum, car le sérum peut empêcher l&#39;absorption de la sonde. </li><li> Ajouter 10 ul d&#39;une SNARF-1 solution 1 mM pour obtenir afconcentration inal de 5 uM et permettent aux cellules incuber pendant 20 min à 37 ° C, sous une lumière faible. C-SNARF-1 et de concentration temps d&#39;incubation peuvent varier en fonction du type de cellule. </li><li> A la fin de l&#39;incubation, laver les cellules deux fois dans du PBS stérile et ajouter 2 ml de milieu sans sérum. </li></ol> 5. Ajout de pH modulateurs <ol><li> Pour évaluer si la méthode peut détecter des changements de pH dans les cellules, les différents réactifs qui modulent le pH intracellulaire peuvent être utilisés: <ul><li> Bafilomycine A1, un inhibiteur spécifique de la V-ATPase 5 </li><li> Na + / H + échangeur isoforme 1 (NHE-1) 6 </li><li> NH 4 Cl, une base faible qui augmente le pH intracellulaire 7 </li></ul><ol><li> Préparer des solutions de des réactifs mentionnés ci-dessus: <ul><li> Bafilomycine A1: ajouter 10 pg de bafilomycine A1 160 ul de DMSO pour obtenir une solution stock de 100 um </li><li> IEAP: ajouter 25 mg de l&#39;IEAP à 1,67 ml de méthanol à obtena concentration finale de 50 mM </li><li> NH 4 Cl: dissoudre 0,53 g de chlorure d&#39;ammonium dans 10 ml de trou DDH 2 O pour obtenir une concentration finale de 1 mM. </li></ul> Remarque: Toutes les solutions mères doivent être conservés à 4 ° C sauf pour bafilomycine A1 qui doit être conservé à -20 ° C et protégé de la lumière. </li><li> Dans des cultures distinctes, ajouter l&#39;un des réactifs tels que décrits ci-dessous: <ul><li> 2 pl de 100 uM bafilomycine A1 (concentration finale de 100 nM) </li><li> 1 pl de l&#39;IEAP 50 mM (concentration finale de 25 uM) </li><li> 40 pl de 1 M de NH 4 Cl (concentration finale 20 mM) </li></ul></li><li> Incuber les cellules pendant 30 min à 37 ° C. </li></ol></li><li> Procéder à l&#39;acquisition de l&#39;image. </li></ol> 6. Spécifications du microscope d&#39;imagerie des cellules vivantes et des paramètres d&#39;acquisition <ol><li> Effectuer imagerie des cellules vivantes en utilisant un objectif 40x monté sur un balayage inversé microsco confocale spectralepe équipé d&#39;une platine XY motorisée, une chambre pouvant accueillir des plats de verre de la culture de fond dans un (température, humidité, CO 2) incubateur à environnement contrôlé de phase. </li><li> L&#39;instrument confocal (Olympus FV1000) utilisé ici est équipé de: <ul><li> Un laser à diode de 405 nm </li><li> A 40 mW laser argon (458 nm, 488 nm, 515 nm) </li><li> Un laser hélium néon (543 nm) </li><li> Objectif fluorite 10X, NA0.4 </li><li> Un objectif LUCPLFLN 40X, NA0.60 </li><li> Un objectif de l&#39;huile PLAPONSC PLAN APO 60X, NA1.4 </li><li> Un miroir dichroïque DM 405/458/488/543 </li><li> Un SDM 560 et un SDM 640 filtres dichroïques </li><li> Un filtre de barrière BA655-755 </li><li> Un BS20/80 de diviseur de faisceau </li><li> logiciel d&#39;analyse de données pour l&#39;acquisition et l&#39;analyse d&#39;images, </li></ul></li><li> Une fois l&#39;incubateur d&#39;étape a réchauffé à 37 ° C et le microscope est mis en place, placer une boîte de culture préalablement incubé avec HHP et SNARF-1 dans la chambre d&#39;imagerie confocale stade. </li><li> Après avoir localiser une zone d&#39;intérêt, régler les paramètres d&#39;acquisition de logiciels Figure 1. <ul><li> Mettre en place deux ensembles de canaux virtuels. </li><li> Initier la première phase qui correspond à HPTS excitation à 405 nm et la collecte d&#39;émission à 505-525 nm et SNARF-1 excitation à 543 nm et la collecte d&#39;émission à 570-600 nm. </li><li> Lancer la deuxième phase qui correspond à HPTS excitation à 458 nm et la collecte d&#39;émission à 505-525 nm et SNARF-1 excitation à 543 nm et la collecte d&#39;émission à 640-650 nm. </li><li> L&#39;ouverture confocale est fixé à 175 um par défaut et l&#39;ouverture optimale doit être réglé manuellement à 300 um. </li><li> Prenez l&#39;image par l&#39;empilement des sections optiques horizontales série (800 x 800 pixels) de 0,4 um d&#39;épaisseur. </li></ul></li></ol> NOTE: Un objectif 40X est utile d&#39;observer plusieurs cellules simultanément et fonctionne bien pour organite visualisation. Cependant, la résolution spatiale et de l&#39;intensité du signal sont better obtenue en utilisant un objectif 60x huile. En outre, il n&#39;y a pas d&#39;excitation ou d&#39;émission de la diaphonie entre les deux colorants. 7. Étalonnage de sondes pH-détection <ol><li> Avant l&#39;étalonnage, éliminer le milieu de la boîte de culture préalablement incubé avec HHP et SNARF-1, laver deux fois avec du PBS stérile, puis ajouter 2 ml d&#39;une des solutions d&#39;étalonnage cinq. </li><li> Placez la boîte de culture dans la chambre de stade confocale. </li><li> Procéder à une acquisition d&#39;image temps-lapse selon les paramètres suivants: <ol><li> Définissez le nombre d&#39;images et de durées d&#39;intervalle pour l&#39;expérience. NOTE: Nous utilisons généralement un intervalle de 1 min entre les images pour un maximum de 30 minutes. </li><li> Programmer le déclencheur pour fermer entre l&#39;acquisition de chaque image. </li></ol></li><li> Enregistrer l&#39;intensité de fluorescence par minute dans un logiciel de feuille de calcul et de déterminer le moment où l&#39;intensité de fluorescence est stable. PH intracellulaire est généralement stable après le 15 - 20 min. </li><li> REPEAT les étapes 7.1 à 7.4 en utilisant une nouvelle boîte de culture pour chaque solution d&#39;étalonnage restant. </li></ol> 8. D&#39;acquisition de données <ol><li> Une fois le calibrage a été fait, l&#39;acquisition de données de l&#39;échantillon 4-5 champs choisis au hasard contenant 15-20 cellules par champ. Deux images sont obtenues pour chaque domaine: l&#39;un pour l&#39;étiquetage HPTS (vésicules) et le second pour SNARF-1 étiquetage (cytosol). </li><li> Fluorescence de fond (zone extracellulaire) est numériquement soustrait des images fluorescentes de deux sondes. Vésicules marquées et cytosol de leurs images stockées respectives sont sélectionnés à l&#39;aide d&#39;un masque binaire. Pour chaque sonde pH-détection, les intensités de fluorescence sont mesurées sur une échelle numérique (0-4,095 échelle de niveaux de gris). Une intensité moyenne de tous les pixels dans le cytoplasme ou organelle est calculée et les valeurs sont utilisées pour calculer les rapports de fluorescence. </li></ol> 9. Analyse des données <ol><li> Divers logiciels sont disponibles dans le commerce pour l&#39;analyse des données. DireFluoview logiciel. </li><li> Dans une feuille de calcul, valeurs de l&#39;enregistrement et le calcul des ratios de fluorescence comme suit: Dans le cas de HPTS, R = (F 458 nm / F 405 nm) et, dans le cas de SNARF-1, R = (F 644 nm / F 584 nm ). </li><li> Tracer les données en utilisant un logiciel graphique scientifique comme GraphPad Prism. La parcelle doit présenter des ratios de fluorescence (axe Y) en fonction du pH (axe X). d&#39;ajustement de courbe est réalisé en utilisant une régression non linéaire. </li><li> Recueillir les intensités de fluorescence des échantillons de la manière décrite dans l&#39;étape 8.2 et de calculer les rapports de fluorescence. Utilisation des courbes d&#39;étalonnage pour transformer en ratios des valeurs de pH. </li></ol>

<ol>
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No conflict of interest declared.

Mesure du pH quantitative en imagerie des cellules vivantes de différents compartiments cellulaires est nécessaire de mesurer avec précision les variations de pH dans les réponses cellulaires aux défis extérieurs. Cependant, l&#39;un des principaux obstacles qui reste est de cibler facilement et précisément compartiments cellulaires. A cet égard, un certain nombre d&#39;études ont mis en évidence l&#39;utilisation de HPTS comme indicateur de pH intracellulaire introduit dans les cellules par électroporation ...

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Solutions for calibration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">D-Glucose (dextrose)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">D16-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Magnesium sulfate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M65-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">HEPES</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Calcium chloride dihydrate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-1612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Nigericin</td>
			<td style="width:136px;">Calbiochem</td>
			<td align="right" style="width:119px;">481990</td>
			<td>Toxic by inhalation or in contact with skin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;">Keep protected from the light at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Culture dishes &#8709; 35mm</td>
			<td style="width:136px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH-sensing probes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">H-348</td>
			<td>Keep away from the light at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">5-(and-6)-Carboxy SNARF&#174;-1, Acetoxymethyl Ester, Acetate</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">C-1271</td>
			<td>Keep away from the light and humidity at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Dimethyl Sufoxide (DMSO)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">BP231-1&#160;</td>
			<td>Harmful</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium phosphate monobasic</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">S369-1</td>
			<td>115 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium phosphate monobasic</td>
			<td style="width:136px;">J.T Baker</td>
			<td style="width:119px;">3246-01</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Autoclave and adjust pH to 7.4</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 1X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">PBS 100X</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>50 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td>40,5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deoinized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH modulators</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">&#160;Bafilomycin A1</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">B-1793</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Ammonium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">A649-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous ph measurement in endocytic and cytosolic compartments in living cells using confocal microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Pour la mesure simultanée du pH intracellulaire dans les deux compartiments de la cellule et endosomal / lysosomal, les sondes fluorescentes pH détection ratiométrique SNARF-1 et HPTS ont été utilisés. SNARF-1 est limitée au compartiment cytosolique tandis HPTS permet la mesure ratiométrique de pH du compartiment endosomal / lysosomal. Cellule de chargement avec HPTS pour 16 heures permet sa localisation sélective dans les endosomes / lysosomes 4. Un exemple typique de fluorescence enregistrée à pa...

Watch this Scientific Journal Video about Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy at JoVE.com

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Mesure simultanée du pH dans endocytose et cytosoliques compartiments dans les cellules vivantes en utilisant la microscopie confocale

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

simultaneous-ph-measurement-endocytic-cytosolic-compartments-living

Research

JoVE Journal

Biology

14.9K vues.  Université de Sherbrooke. Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells. 

Vidéo: Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Changement de forme d&#39;imagerie cellulaire en vie Drosophile Embryons

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

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Biologie

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basées sur la fluorescence de mesure de l&#39;entrée du calcium magasin fonctionnant dans des cellules vivantes: à partir de cellules cancéreuses en culture de fibre musculaire squelettique

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Mesure du pH vacuolaire et cytosolique<em&gt; In Vivo</em&gt; En cellule de levure Suspensions

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analyser craniofaciale morphogenèse chez le poisson zèbre Utilisation de 4D microscopie confocale

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo suivi clonale de cellules souches hématopoïétiques et progénitrices Marqué par cinq protéines fluorescentes confocale et multiphotonique Microscopie

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Mesures de calcium cytosolique dans les cellules épithéliales rénales par cytométrie en flux

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Mesure simultanée de mitochondrial Calcium et mitochondrial potentiel membranaire dans des cellules vivantes par microscopie à fluorescence

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Sonder la cinétique de l’ARNm dans l’espace et le temps à Escherichia coli à l’aide de l’hybridation de fluorescence in situ à deux couleurs

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...