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.

Hücresel pH Kalibrasyon Çözümleri 1. Hazırlanması <ol><li> Kalibrasyon için kullanılmak üzere 5 çözümler hazırlamak için beş ayrı 50 ml konik tüplere aşağıdaki bileşikler ekleyin: <ul><li> NaCl (1 M NaCl 1 mi) (1 M NaCl, 10 ml H = 2 0 içinde 0.58 g) </li><li> KCl (6.75 1 M KCI mi) (1 M KCl, 10 ml H 2 0 içinde 0.75 g =) </li><li> Glikoz (1 M D-Glikoz 1 mi) (1 M D-Glucose = H 10 ml 1.80 g 2 0) </li><li> MgS0 4 (0.05 1 M MgSO 4 ml) (1 M, 10 ml MgSO H 2 0 içinde 4 = 1.20 g) </li><li> 20 mM HEPES (1 M HEPES 1 mi) (10 mi H 2 0 içinde 1 M HEPES = 2.38 g) </li><li> CaCl2 (1 M CaCI2 ve 0.05 mi) (10 mi H 1 M CaCl2 = 1.47 g 2 0) Çift distile H 2 O (GKD 2 O) tüm çözümler hazırlayın. </li></ul></li><li> GKD 2 O 35 ml solüsyonu ile, her tamamlamak ve böylece kadar bir manyetik karıştırıcı ile karıştırındevrim sisi elde edilir. </li><li> 5.5, 6.0, 6.5, 7.0 ve 7.5 pH değeri elde etmek üzere 1 N KOH ya da 1 N HCI ile, her çözeltinin pH&#39;ı ayarlayın. Daha sonra, 50 ml lik bir son hacme kadar deiyonize su ile hacim ayarlayın. </li><li> Tedbir 5 nigerisin mg ve stok çözeltisi nigericin 10 mm hazırlamak için mutlak EtOH 670 ul ekleyin. Dikkat: nigericin son derece toksik ve büyük bir dikkatle (eldiven, gözlük ve maske) ile ele alınması gerekir. Nigericin tamamen eriyene kadar inversiyon yolu ile çözüm karıştırın. </li><li> Her bir ayar çözeltiye 10 mM nigerisin (nihai konsantrasyon 10 uM) ve 0.05 ml ilave edilir. Nigericin hücre zar geçirgenliğini artırır ve hücre dışı K + kutuplaşma giderici bir konsantrasyonunun mevcudiyetinde hücre-içi ve hücre dışı pH&#39;ın dengede neden olan bir iyonofor olduğunu. </li><li> Kalibrasyon çözümleri 4 ° C&#39;de 1 ay kadar saklanabilir </li></ol> 2.. Hücre Hazırlanması <ol><li> Biyolojik bir Safet altında35 mm y kapağı, tohum hücreleri x 10 mm kültür tabakları,% 10 fetal sığır serumu ve antibiyotikler ile desteklenmiş büyüme ortamı içinde 2 ml. 24 saat sonra yaklaşık olarak% 50 izdiham sağlamak için gerekli hücre miktarı kullanın. Not: Deneylerde, biz HT-1080 hücre çizgisi ve plaka kültür kabı başına 1 x 10 5 hücre kullanın. </li><li> Kalibrasyon için kullanılacak deney ve plaka 5 ek kültür tabakları için gereken kültür kaplarına sayısını belirler. </li><li> En az 24 saat boyunca 37 ° C /% 5 CO2 inkübatörü içinde yer kültür tabakları. </li></ol> PH-algılama Sondalar 3. Hazırlanması <ol><li> GKD 2 0 floresan prob pH 8-hydroxypyrene-1 ,3,6-trisülfonik asit, trisodyum tuzu (HPTS / pyranine) bir 1 M stok çözelti hazırlayın. Bu çözelti, oda sıcaklığında saklanan ve ışıktan korunmalıdır. </li><li> Thereâ adlandırılan (and-6)-karboksil seminaphthorhodafluor asetoksimetil ester asetat (C-SNARF-01:00 - 1 mM 5 &#39;lik bir stok çözelti hazırlayınfter SNARF-1) DMSO 1.76 ml Snarf-1, 1 mg çözülmesiyle. Iyice tekrarlanan pipetleme karıştırın. SNARF-1 çözeltisi, -20 ° C&#39;de saklanan ve ışıktan korunmalıdır. </li></ol> 4. Hücre Etiketleme <ol><li> Onaltı saat canlı hücre görüntüleme önce, tam ortam 2 ml içeren her kültür tabağına 1M HPTS stok solüsyonu 2 ul (nihai konsantrasyon 1 mM) ekleyin. </li><li> Inkübatör kültür yemekleri dönün. HPTS prob nedenle etiket prob pinositoza tarafından alınmalıdır organeller, hücre-laktobionat olduğunu. Bu durum HPTS hücre inkübasyonlar serum-takviyeli kültür ortamında gerçekleştirilir gerektirir. </li><li> HPTS ile inkübasyondan sonra, steril PBS ile iki kez Hücreleri yıkamak ve en az 2 saat boyunca serumsuz ortamda 2 ml ekleyin. SNARF-1 ile inkübasyon serum prob ortaya çıkmasını önlemek, çünkü serum içermeyen ortam içinde yapılması gerekmektedir. </li><li> AF elde etmek üzere bir 1 mM SNARF-1 ihtiyat çözeltisinden 10 ul ekleinal 5 uM arasında konsantrasyon ve hücreler loş ışık altında, 37 ° C&#39;de 20 dakika boyunca inkübasyona izin verin. C-SNARF-1 konsantrasyon ve kuluçkalama süresi, hücre tipine bağlı olarak değişebilir. </li><li> İnkübasyonun sonunda, steril PBS içerisinde iki kez yıkama ve hücreler serum içermeyen ortam 2 ml ekleyin. </li></ol> PH Modülatörler 5. Ilavesi <ol><li> Yöntem hücrelerde pH değişiklikleri tespit edebilir olup olmadığını değerlendirmek için, hücre içi pH&#39;ın modüle farklı reaktif de kullanılabilir: <ul><li> Bafilomisin A1, V-ATPaz 5 spesifik bir önleyicisi </li><li> Na + / H + değiştirici izoformu 1 (NHE-1) 6 </li><li> NH4CI, hücre içi pH 7 arttıran bir zayıf baz </li></ul><ol><li> Yukarıda bahsedilen reaktiflerin stok çözelti hazırlayın: <ul><li> Bafilomisin A1: 100 uM&#39;lik bir stok çözeltisi elde etmek üzere, DMSO ile 160 ul bafilomycin A1 10 ug eklemek </li><li> EIPA: obtai metanolün 1.67 ml EIPA 25 mg eklemek50 mM&#39;lik nihai konsantrasyon na </li><li> NH4CI: 1 mM&#39;lik bir son konsantrasyon elde etmek üzere GKD 2 O 10 ml amonyum klorürün 0.53 g çözülür. </li></ul> Not: Tüm stok solüsyonları -20 ° C sıcaklıkta saklanan ve ışıktan korunmalıdır bafilomycin A1 dışında 4 ° C&#39;de tutulmalıdır. </li><li> Ayrı bir kültür tabaklarında, aşağıda tarif edildiği gibi reaktifler ekleyin: <ul><li> 100 uM bafilomycin A1 2 ul (son konsantrasyon 100 nM) </li><li> 50 mM EIPA 1 ul (nihai konsantrasyon 25 uM) </li><li> 1 M NH4CI (nihai konsantrasyon 20 mM), 40 ul </li></ul></li><li> 37 ° C&#39;de 30 dakika boyunca inkübe hücreleri </li></ol></li><li> Görüntü alma geçin. </li></ol> 6.. Canlı hücre Görüntüleme Mikroskop Özellikleri ve Toplama Ayarlar <ol><li> Kullanarak canlı hücre görüntüleme gerçekleştirmek 40X objektif bir spektral ters tarama konfokal microsco üzerine montepe motorize XY aşamada, çevre kontrollü (sıcaklık, nem, CO 2) evre inkübatör cam alt kültür yemekleri barındıran bir bölme ile donatılmıştır. </li><li> Burada kullanılan konfokal alet (Olympus FV1000) ile donatılmıştır: <ul><li> Bir diyot lazer 405 nm </li><li> A 40 mW argon lazer (458 nm, 488 nm, 515 nm) </li><li> Bir helyum neon lazer (543 nm) </li><li> Florit 10X objektif, NA0.4 </li><li> Bir LUCPLFLN 40X objektif, NA0.60 </li><li> Bir PLAPONSC PLANI APO 60X yağ objektif, NA1.4 </li><li> Dikroik ayna DM 405/458/488/543 </li><li> Bir SDM 560 ve SDM 640 dikroik filtreleri </li><li> A BA655-755 bariyer filtre </li><li> Bir ışın ayırıcı BS20/80 </li><li> Görüntü elde etme ve analiz için veri analizi yazılımı, </li></ul></li><li> Sahne inkübatör 37 ° C&#39;ye kadar ısıtılır ve mikroskop kurulduktan sonra, önce konfokal sahne görüntüleme odasına HPTS ve Snarf-1 ile inkübe bir kültür çanak yerleştirin. </li><li> Ailgi alanı bulma ürkiye&#39;de, yazılım satın alma ayarları Şekil 1 ayarlayın. <ul><li> İki sanal kanal set kurmak. </li><li> 525 nm ve 570 nm 543 ve emisyon toplama SNARF-1 uyarım - - 600 nm 505 405 nm ve emisyon toplama HPTS uyarma karşılık ilk aşamasını başlatın. </li><li> 525 nm ve 640 de 543 nm ve emisyon toplama SNARF-1 uyarım - - 650 nm 505 458 nm ve emisyon toplama HPTS uyarma karşılık ikinci aşamasına. </li><li> Konfokal açıklık varsayılan olarak 175 um ayarlanır ve optimal diyafram elle 300 mikron ayarlanmış olması gerekiyor. </li><li> 0,4 mikron kalınlığında seri yatay optik bölümleri (800 x 800 piksel) istifleme Resmi atın. </li></ul></li></ol> NOT: 40X objektif aynı anda birkaç hücreleri gözlemlemek için yararlıdır ve organel görüntüleme için iyi çalışır. Ancak, uzaysal çözünürlüğü ve sinyal yoğunluğu Bette vardırR, bir 60X objektif kullanılarak yağ elde. Buna ek olarak, iki boyalar arasında bir uyarım veya emisyon cross-talk bulunmaktadır. PH-algılama Problar 7. Kalibrasyon <ol><li> Kalibrasyon önce, daha önce HPTS ve Snarf-1 ile inkübe kültür çanak orta kaldırmak steril PBS ile iki kez yıkama ve daha sonra, beş kalibrasyon çözümlerden biri 2 ml ekleyin. </li><li> Konfokal aşama odasına kültür tabağına yerleştirin. </li><li> Aşağıdaki ayarlara göre zaman atlamalı görüntü elde geçin: <ol><li> Deney için görüntülerin ve aralık süresi sayısını ayarlayın. NOT: tipik olarak 30 dakika ila görüntüleri arasında, 1 dakika süre aralığı kullanınız. </li><li> Her görüntünün edinimi arasındaki kapatmak için deklanşörü programlayın. </li></ol></li><li> Bir elektronik tablo yazılımı dakika başına floresan yoğunluğunu kaydedin ve floresan yoğunluğu sabit olduğu zaman belirler. 20 dk - Hücre içi pH 15 sonra genellikle kararlı. </li><li> REPEAT kalan her kalibrasyon çözümü için yeni bir kültür çanak kullanarak 7,1-7,4 adımları. </li></ol> 8. Veri Toplama <ol><li> Kalibrasyon yapıldıktan sonra, alan başına 15-20 hücreleri içeren 4-5 rastgele seçtikleri alanlarda örnek verileri kazanır. İki görüntüleri her alan için elde edilir: HPTS etiketleme (kesecikler) ve SNARF-1 etiketleme (Sitosol) için saniye biri. </li><li> Eşiğe (hücre dışı bir bölge) dijital hem de sondaların floresan görüntülerden çıkarılır. Ilgili saklanan görüntülerden Etiketli veziküller ve sitosol bir ikili maske kullanılarak seçilir. Her pH-algılama probu için, floresan yoğunlukları sayısal bir ölçek (0-4,095 gri seviye ölçeği) ölçülür. Organel ya da sitoplazmada tüm pikseller için bir ortalama yoğunluğu hesaplanır ve değerler floresan oranlarının hesaplanması için kullanılmaktadır. </li></ol> 9. Veri Analizi <ol><li> Çeşitli yazılım paketleri veri analizi için piyasada mevcuttur. YaniFluoview Yazılım. </li><li> Aşağıdaki gibi, bir tablo, kayıt değerleri içinde ve floresan oranları hesaplanır: HPTS durumunda, R = (F 458 nm / F 405 nm), ve SNARF-1, R = (F 644 nm / F 584 nm durumunda .) </li><li> Böyle GraphPadPrizmasındaki gibi bilimsel grafik yazılımı kullanarak veri çiziniz. Plot pH (X ekseni) bir fonksiyonu olarak floresans oranı (Y-ekseni) tasvir gerekir. Eğri uydurma, lineer olmayan regresyon kullanılarak elde edilir. </li><li> Adım 8.2 &#39;de tarif edildiği gibi örneklerin floresan şiddetlerini toplamak ve floresan oranları hesaplar. PH değerleri içine oranlarını dönüştürmek için kalibrasyon eğrileri kullanın. </li></ol>

<ol>
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No conflict of interest declared.

Farklı hücre bölümlerinde canlı hücre görüntüleme Sayısal pH ölçümü doğru dış zorluklara hücre yanıtlarında pH değişimleri ölçmek için gereklidir. Bununla birlikte, hala önemli engellerden biri kolayca ve özel hücre bölmeleri hedef etmektir. Bu bağlamda, bir kaç çalışmada, elektroporasyon ya da mikroenjeksiyon ile 8-10 hücrelere katılan bir hücre içi pH göstergesi olarak HPTS kullanımını sermiştir. Bu teknikler yani elektro hücrelere geniş zararlara yol açar, ve m...

<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.

Hücre içi ve endozomal / lızozomal hücre bölmelerine hem de pH değerinin ölçümü için aynı anda, rasyometrik floresan pH algılama Snarf-1 problar ve HPTS kullanılmıştır. HPTS endozomal / lizozom oran ölçer pH&#39;ı ölçümünü sağlar, oysa SNARF-1 sitosolik bölmesi ile sınırlandırılmıştır. 16 saat HPTS ile hücre yükleme endozomlar / lizozomlarında 4 seçici yerelleştirme sağlar. PH algılama probları ile yüklü HT-1080 hücreleri kaydedilen floresans tipik bir örneği, <s...

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.

Konfokal mikroskopi kullanılarak Yaşayan hücreler içinde endositik ve Sitosolik Bölümleri eşzamanlı pH Ölçümü

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 Görüntüleme.  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. 

Video: 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.

Living Görüntüleme Hücre Şekli Değiştir Drosophila Embriyolar

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

Biyoloji

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.

Canlı hücreler Mağaza çalışan Kalsiyum Giriş Floresans tabanlı Ölçümü: Kültür Kanseri Hücre gelen İskelet Kası Fiber

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).

Vakuolar ve sitosolik pH Ölçümü<em&gt; In Vivo</em&gt; Maya hücre süspansiyonlarında

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.

4D konfokal mikroskopi kullanılarak Zebra balığı Kranyofasiyel morfogenez Analizi

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.

Eşodaklı ve multiphoton Mikroskopi kullanarak beş Floresan Proteinleri ile işaretlenmiş Hematopoetik kök hücrelerin in vivo klonal Takip

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.

Sitometrisi ile Böbrek Hücrelerine sitozolik kalsiyum Ölçümleri

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.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

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.

Floresan Mikroskopi Canlı hücreler Potansiyel Mitokondriyal Kalsiyum ve Mitokondriyal Membran Eşzamanlı Ölçümü

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.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

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

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Toplam İç Yansıma Floresan Mikroskobu Ile Canlı Hücrelerdeki Hücre Yüzey Reseptörlerinin Oligomerizasyon Dinamiği Sayı ve Parlaklık Analizi ile Birlikte

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...