Bu çalışma PM Kane için NIH R01 GM50322 tarafından desteklenmiştir. Yazarlar bizim laboratuar için bu protokollerin çalışma dışarı Dr Rajini Rao, maya pHluorin plazmid sağlamak için ve rasyometrik pH ölçümleri tavsiye için Johns Hopkins Üniversitesi ve Dr Gloria A. Martinez Munoz teşekkür ederim.

1. BCECF-AM kullanarak in vivo Vakuolar pH Ölçümü <ol><li> Gece boyunca istenen ortam ölçülecek olan maya suşu, 50 ml sıvı kültür büyütün. Amaç, (OD600 (600 nm&#39;de optik yoğunluk) süspansiyon yaklaşık 0.8 ölçümü) orta log fazında hücrelerin sahip olmaktır. </li><li> Pelet santrifüj maya hücreleri. Büyüme ortamı ve önceden tartılan edilmiş bir mikrosantrifüj tüpüne transfer 0.6 ml pelletini. 60 saniye boyunca 2000 x g&#39;de bir mikrosantrifüj içinde yeniden Pelet hücreleri. Olarak tamamen mümkün süpernatant kaldırmak ve daha sonra hücre pelet tartmak. 0.5 g / ml (k / h) bir son yoğunluk elde etmek için pelletini, bu birimin bir kültür, yaklaşık olarak 200 mg arasında hücre topağı, ve tamponun 200 ul verecek 400 ul&#39;lik nihai bir hacim verecek şekilde eklenebilir. </li><li> DMSO içinde hazırlanan 12 mM stoktan 50 mM&#39;lik bir son konsantrasyonda hücre süspansiyonu BCECF-AM ekleyin. Iyi ve daha sonra inc Mix30 dakika boyunca 30 ° C&#39;de hücre Ubate. bir sallanan bir platform veya rulo davul. </li><li> Hücreler inkübe edilirken, kalibrasyon tamponlar hazırlamak. , 50 mM HEPES (4 - (2-hidroksietil)-1-piperazinetansülfonik asit), 50 mM KCI, 50 mM NaCI, 0.2 M amonyum asetat, - kalibrasyon tamponu 50 mM MES ((N-morfolino) etansülfonik asit, 2) içerir 10 mM sodyum azit ve 10 mM 2-deoksiglukoz, ve pH değeri ayarlanır NaOH veya HCI ile kalibrasyon aralığı için uygun değer verir. (Dikkat: Sodyum azid yüksek derecede toksiktir ve dikkatle ele alınmalıdır.) Yabani tip hücrelerde pH ölçümü için, (6.5, ama bir daha alkali vakuolar pH olması bekleniyor mutantlar için pH 5.5 &#39;de birkaç kalibrasyon karışımları hazırlayacak vma mutantlar), ek, daha yüksek pH tamponları hazırlayacak. </li><li> Kısım 2 15 ml konik tüpler her bir pH için kalibrasyon tampon ml, ve son 110 mcM monensin konsantrasyonu ve 15 mcM nigericin, r vermek için monensin (15 mM stok) ve nigericin (2 mM stok) ekleyinespectively. Bir vorteks karıştırıcıda iyice karıştırın. (Dikkat: monensin ve nigericin Hem toksik ve dikkatle ele alınmalıdır.) </li><li> bir mikrosantrifüj içinde 2,000 x g&#39;de 30 saniye boyunca santrifüj ile BCECF-AM inkübasyon süresi tamamlandıktan sonra, pelet hücre süspansiyonu. Hücrelerin tekrar yukarıdaki gibi santrifüj eksik glukoz ve büyüme ortamı 1 ml&#39;dir. Bir kez bu yıkama adımı yineleyin ve sonra glikoz olmadan büyüme orta 200 ul pelet tekrar süspansiyon haline getirin. Buz üzerinde yer. </li><li> Yukarıda hazırlanan her bir pH kalibrasyon tüp hücre süspansiyonu, 20 ul ekle. 30-60 dakika boyunca 30 ° C&#39;de inkübe edin. bir rulo davul. </li><li> Dönüşümlü olarak uyarım dalga boyu 450 nm ve 490 nm, 535 nm ve emisyon dalga boyu ile hem de ölçmek için florimetresi ayarlayın. 30 ° C için örnek odası sıcaklığı ayarlayın Küvet içinde, karışımın sürekli karıştırma ile tüm ölçümler gerçekleştirme. </li><li> 1 mM MES ve 1.96 ml (hücrelerin büyüme ortamı arasında bir pH değerine bağlı olarak pH değeri 5 ya da 7&#39;ye ayarlandı eklemend deneysel tasarım). Küvet içine hücre süspansiyonu, 20 ul ekle. Floresan ölçümleri başlatın. Biz sürekli kinetik ve farklı deneylerde zaman noktası veri hem toplamak. Sürekli kinetik veriler için, 5 dakika için 6 saniye ölçüm almak, sonra 50 mM&#39;lik bir son glükoz konsantrasyonu için glikoz ekleyin ve 5-10 dakika için ölçü devam etmektedir. Tek zaman noktası ölçümleri için, biz genel olarak 1 ölçümleri almak ve 5 dk. küvete hücrelerin eklenmesinden sonra, kinetik ölçümler olarak glikoz eklenmektedir ve daha sonra başka bir ölçüm 5 dakika sürer. glukoz ilave edildikten sonra. Bu eklemeler değişik olabilir ve ek bileşenler (inhibitörleri, vs) de ilave edilebilir. </li><li> Deneysel ölçümler tamamlandıktan sonra, 30 ° C&#39;de kuluçka, kalibrasyon tüpleri kaldırmak ve florimetresi küvete her biri için tüm 2 ml hacme aktarın. Her 5 sn her bir örnek için 30 sn olmak üzere toplam üzerinden (1.8 bakınız) aynı ayarlarda floresan ölçün. </li><li>Microsoft Excel&#39;e floresan İhracat verileri. (Bizim florimetresi için, bu Excel&#39;e sekme ile sınırlandırılmış formu ve ithalat metin olarak veri ihracat gerektirir.) Her kalibrasyon karışım için 450 nm 490 nm floresan oranı hesaplayarak bir kalibrasyon eğrisi elde. Floresans oranı, daha sonra, bir kalibrasyon eğrisi elde etmek için pH değeri genel çizilir. </li><li> Floresan oranı deneysel verileri dönüştürmek ve standart eğrisi kullanılarak pH hesaplar. Vakuolar sonra pH süresi (pH değişimi kinetiği) karşı çizilen veya çeşitli koşulları (Şekil 1) altına ya da farklı mutant suşları karşılaştırıldığında olabilir. </li></ol> 2. Maya pHluorin kullanarak in vivo Sitosolik pH Ölçümü <ol><li> Standart protokoller plazmid maya pHluorin ile istenilen maya suşu dönüştürmek ve takviye az orta eksik urasil (SC-urasil) üzerinde transformantlar için seçin. </li><li> Orta log fazı (OD SC-urasil dönüştürülmüş hücre, 50 ml sıvı kültür büyütün <sub&gt; 600 = 0.8 veya daha düşük). </li><li> Vakuolar pH, ancak sitosolik pH ölçümleri, genellikle pH 6-8 için uygun bir pH tampon için tarif edilen kalibrasyon standardı hazırlayın. Kalibrasyon tampon kısım 2 mi birkaç 15 ml konik tüp içine istenen pH değerine ayarlandı. (1.4) yukarıda tarif edildiği gibi Monensin ve nigericin ekleyin ve iyice karıştırın. </li><li> Yukarıda tarif edildiği gibi santrifüj ile hücreler hasat ve büyüme ortamı 600 ul pelletini. 30 saniye için 5000 rpm&#39;de santrifüjleme ile tartılmış bir mikrosantrifüj tüpü ve topak hücrelere transfer. Yine glikoz (SC-urasil-glikoz), topak hücrelere olmadan büyüme ortamı içinde 1 ml pelletini ve tekrarlayın. Santrifüj işleminden sonra son olarak tamamen mümkün süpernatantı ve hücre pelletini tartın. Resim glukoz ile SC-urasil, 0.5 g / ml &#39;lik bir nihai yoğunluğa süspansiyon hücreleri. </li><li> Kalibrasyon tampon her tüpe hücre süspansiyonu, 20 ul ekleyin ve bir vorteks karıştırıcıda iyice karıştırın. 3 inkübe0 ° 60 dakika için dönen bir davul rotor üzerinde C. </li><li> Dalga boyları 405 ve 485 nm eksitasyon ve 508 nm emisyon dalga boyu için florimetresi ayarlayın. BCECF ölçümü için yukarıda tarif edildiği gibi tek bir zaman noktasında veya kinetik ölçümleri ve glikoz ilave devam edin. </li><li> Kalibrasyon numuneleri arasında floresan ölçmek ve BCECF (1.10-1.11 bakınız) gibi bir kalibrasyon eğrisi oluşturmak. Floresans oranı genel pH Bir arsa en sitosolik pH ölçümleri (pH 6.0-8.0) 9 aralığında doğrusaldır, bu nedenle deneysel floresans oranı ölçümleri pH&#39;ını kolayca dönüştürülür. </li></ol>

<ol>
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Q., 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=Requirement+for+ergosterol+in+V-ATPase+function+underlies+antifungal+activity+of+azole+drugs.">Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs.</a> PLoS Pathog. 6, e1000939 (2010).</li><li>Brett, C. L., 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=Genome-wide+analysis+reveals+the+vacuolar+pH-stat+of+Saccharomyces+cerevisiae.">Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae.</a> PLoS One. 6, e17619 (2011).</li><li>Roberts, C. J., Raymond, C. K., Yamashiro, C. T., Stevens, T. 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Yazarlar ifşa etmek hiçbir çıkar çatışması var.

Biz pH dengesinin yönlerini bir dizi adrese bu protokolleri kullanmış olurlar. Örneğin, 4,5-yabani tip ve V-ATPaz eksikliği mutant hücreler sitozolik pH ve yanıtlar bakımından karşılaştırdık. Ayrıca glikoz 3 vakuolar pH yanıt değişmiş büyüme koşulları, özellikle hücre dışı pH, etkilerini inceledik. Önemlisi, biz gözlemlemek tepkiler hem nicel pH ölçümü diğer yöntemlerle ve proton pompa değişmiş faaliyetleri anlatan biyokimyasal verilerle uyumludur. <p cla...

pH dengesini bütün organizmalar 1,2 dinamik ve oldukça düzenli bir süreçtir. Biyokimyasal süreçlerin sıkı pH tarafından düzenlenir ve hücre içi ortamlarda ikamet enzimlerin en iyi etkinlik sağlamak için dar pH aralıkları için ayarlanmıştır. Ancak, hücre içi pH dengesini çevre pH, metabolik değişimler ve bazı sinyal yolları hızlı değişiklikler itiraz edilebilir. Buna ek olarak, hücre içi pH önemli bir sinyal olarak kendini hizmet edebilir. Son olarak, birçok organeller organel-özel işlevler için çevredeki sitoplazmada farklı ve gerekli olan lumenal pH değerlerini korumak. Yüksek ökaryotlar 2 ile pH dengesini mekanizmaların maya Saccharomyces cerevisiae hisse bir dizi. Lizozomal / endositik yolunun asidik organeller olarak, pH öncelikle birçok exchan ile birlikte hareket eden, son derece korunmuş vakuolar proton-translocating ATPaz (V-ATPaz) tarafından kontrol edilirpH degrade bağlıdır Gers. Tüm ökaryotik hücreler aynı zamanda proton verme mekanizması vardır. Olarak mantar ve bitkiler, plazma zarı ikinci bir, ayrı proton pompa, Pma1, ihracat metabolik proton ve sitozolik pH ve plazma membran potansiyeli en önemli belirleyici olduğuna inanılıyor. S. genetik esnekliği cerevisiae ve ticari önemi, bu pH dengesini 2 eğitim için çok ilginç ve önemli bir model yaptık. Organel asitlenme birincil sürücüleri olmasının yanı sıra, V-ATPaz yüksek enzimler düzenlenir ve laboratuvar V-ATPaz düzenleme mekanizmaları anlamak ilgileniyor. Bu hedefe doğru, biz vakuolar ve sitozolik pH in vivo pH ölçümlerinde kullanıyorum: 1) mutasyonların etkilerini bu uzlaşma V-ATPaz aktivitesini incelemek için glikoz yoksunluk ve readdition, 2 olarak değişen hücre dışı koşullar,) yanıtları izlemek için, ve 3) koordine keşfetmek içinorganel ve plazma membran proton pompa 3-5 koordi-nasyonunu sağlıyor. Bu deneyler sadece maya hücreleri kullanmak için uygun sağlam rasyometrik pH göstergelerin geliştirilmesi yoluyla mümkün oldu. . Memeli hücrelerinde sitosolik pH&#39;ını ölçmek için yaygın olarak kullanılmaktadır,, maya vakuol birikir - Bitki ve ark olanlar BCECF ((ve 6)-karboksifloresan 2&#39;7&#39;-Bis-(2-karboksietil) -5) gösterdi yerine sitoplazmada 6. BCECF lokalizasyon bu fark BCECF-AM (BCECF arasında asetoksimetil ester) ve vaküler tutma 6 asetoksi metil esterin parçalanması için olası sorumludur vakuol birçok hidrolitik enzimler, atfedilmiştir. Ali ve diğ. 7 daha BCECF kullanılarak vakuolar pH ölçümü geliştirilen ve bir floresan plaka okuyucu biçimi için bu ölçümler uyarlanmıştır. Brett ve ark. Bir plazmid kaynaklı R ifade ederek maya sitosolik pH ölçümü için bir araç olarak maya pHluorin tanıtılanBir mantar-spesifik promotör 9 arasında kontrol altında atiometric pH duyarlı GFP 8. BCECF ve maya pHluorin iki uyarma spektrumları pH&#39;a duyarlıdır, bu nedenle tek bir emisyon dalga boyunda ölçülen iki uyarma dalga boyu, önceki floresans oranı pH 8,10 arasında bir ölçü sağlar ki rasyometrik pH göstergesi olarak kullanılır. Bu maya vakuolar ve sitozolik pH sensörleri hem tek hücreli ve toplum temelli ölçümler için kullanılmaktadır. Tek hücreli ölçümleri 6,11 floresan mikroskopi ve görüntü analizi ile gerçekleştirilir. Iki dalga boyunda Vakuolar veya hücre floresan her bir hücre için ölçülür. Popülasyon tabanlı ölçümleri uygun floresan özelliklerine sahip bir mikroplaka okuyucu ya da ya da bir fluorimetre gerçekleştirilir. Bu con sırasında glikoz gibi bileşenlerin yanı sıra kolay erişim sağlar çünkü genellikle, bir florimetresi bizim ölçümleri yapmışmanın hikayesi kinetik ölçümleri. Vakuolar ve sitosolik pH değerinin ölçümü için mevcut laboratuar protokoller aşağıda belirtilmiştir; hem de kolayca mikroplaka deneyleri için uyarlanmıştır.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Spectrofluorometer</td>
 <td>Horiba Jobin Yvon</td>
 <td>Model Fluoromax-4</td>
 <td>Temperature control and stirring capability are desirable.</td>
 </tr>
 <tr>
 <td>BCECF-AM</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>B1150</td>
 <td>Prepare a 12 mM stock in dry DMSO, store as aliquots at -20 &deg;C </td>
 </tr>
 <tr>
 <td>monensin</td>
 <td>Sigma</td>
 <td>M5273</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>nigericin</td>
 <td>Sigma</td>
 <td>N7143</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>MES</td>
 <td>Sigma</td>
 <td>M8250</td>
 <td></td>
 </tr>
 </tbody>
</table>

measurement of vacuolar and cytosolic ph in vivo in yeast cell suspensions

Vakuolar ve pH sitozolik yüksek maya hücreleri düzenlenmiştir ve genel pH dengesini merkezi bir rol işgal edilir. Biz vakuol veya sitoplazmada lokalize pH duyarlı fluorophores kullanarak in vivo pH oranlı metrik ölçümü için protokolleri tarif. Vakuolar pH onun asetoksimetil ester halinde hücreler içine dahil edildikleri zaman, maya vakuol lokalize BCECF ile ölçülmektedir. Sitosolik pH değeri, bir maya promotör, maya pHluorin kontrolü altında ifade edilen, pH&#39;ye duyarlı, GFP ile ölçülür. Bir fluorimetre maya hücre süspansiyonlarında floresan oranları ölçümü için yöntemler tarif edilmiştir. Bu protokollerin sayesinde, farklı koşullar altında ya da farklı maya mutant pH tek bir zaman noktasında ölçümleri karşılaştırılmıştır ve zaman içinde pH değişiklikleri takip edilmiştir. Bu yöntemler aynı zamanda yüksek hacimli deneyler için flöresanlı bir plaka okuyucu biçimine uyarlanmıştır. Diğer ap üzerinde rasyometrik pH ölçümleri Avantajlarışu anda kullanılan, olası deneysel sorunlar ve çözümleri ve bu tekniklerin ileride kullanmak için umutları izlenen yolun da açıklanmıştır.

50 mM MES pH 5&#39;e tamponlu: Şekil 1 zengin bir ortamda yetiştirilen yabani tip maya hücreleri (YEPD maya ekstresi, pepton-dekstran) üzerinde elde vakuolar pH veriler sunulmuştur. Ortamın pH özellikle minimal ortam için gece büyüme sırasında oldukça önemli ölçüde değişebilir, çünkü genellikle tamponlu ortam içinde hücrelerin büyümesi, ve büyüme ortamının pH&#39;ı, pH vakuolar yanıtları 3 etkileyebilir bulduk. Çok sayıda deney tamponsuz ortamda hücrelerin ...

Watch this Scientific Journal Video about Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions at JoVE.com

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

Vakuolar ve sitozolik pH canlı maya ölçülebilir (<em&gt; S. cerevisiae</em&gt;) Özel hücre bölmeleri lokalize rasyometrik floresan boyalar kullanarak hücreleri. Biz maya vakuol için lokalize BCECF-AM, ve bir sitozolik rasyometrik pH duyarlı GFP (maya pHluorin) ile sitozolik pH ile vakuolar pH ölçümü için prosedürler tarif.

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 ...

measurement-vacuolar-cytosolic-ph-vivo-yeast-cell

Research

JoVE Journal

Biology

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

19.8K Görüntüleme.  SUNY Upstate Medical University. Vakuolar ve sitozolik pH canlı maya ölçülebilir ( S. cerevisiae) Özel hücre bölmeleri lokalize rasyometrik floresan boyalar kullanarak hücreleri. Biz maya vakuol için lokalize BCECF-AM, ve bir sitozolik rasyometrik pH duyarlı GFP (maya pHluorin) ile sitozolik pH ile vakuolar pH ölçümü için prosedürler tarif.

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

İskelet kas-içi hayati Mikroskopi in vivo Mikro sirkülasyon Ölçümü

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Biyoloji

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Tek hücreli fare Koni fotoreseptörlerin Emiş Kayıtlar

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Tek ve Çok hücreli Bağlamlarda Invadopodia aracılı Ekstrasellüler Matriks Proteoliz Kantitatif Ölçümü

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Testis Hücre Cezalı Hazırlanması için basit ve Verimli Tekniği

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

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.

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 ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

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.

Spektral Sitometrisi ile Katı Dokular İzole Hücre Cezalar Analizi

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 ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Yüksek çözünürlüklü görüntüleme ve Bireysel Astral Mikrotubul Dynamics Analizi Budding Maya

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

Maya 4D mikroskopisi

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Floresan proteinlerin etkisi Polyglutamine toksisite deneyleri Maya kullanılarak füzyon ortakları

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Vakuolar ve sitosolik pH Ölçümü

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