We thank J. Schmidt from the University of Freiburg for constructing the device. We thank P. Nielsen and D. Medgyesi for their support and critical reading of this manuscript. This study was supported by the Deutsche Forschungsgemeinschaft through SFB746 and by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

1. Tasarımı ve Aygıtı Bina <ol><li> Pilot Deneyler NOT: Belirli bir optogenetic aracı ve hücre tipi için gerekli olan ışık yoğunluğu önemli ölçüde değişebilir. prototip Pilot deneyler gerekli minimum ışık yoğunluğunu tahmin etmek yararlıdır. Aşağıdaki deneyler için kullanılan Optogenetic aracı Dronpa-Bağlayıcı-Dronpa füzyon konstruktu olup. Dronpa dizisi ticari (Malzeme Listesi) elde edildi. Uzun bir bağlayıcı ifade yapısı, molekül-içi (ve moleküller arası) dimerlerinin oluşturulması için izin vermek için iki Dronpa diziler arasında klonlandı. aşağıda tarif edilen kademeler bir çok Optogenetic araçları veya optik olarak kontrol edilen maddelere adapte edilebilir. <ol><li> ambalaj hücrelerden retroviral-ihtiva eden üst sıvıyı kullanarak transdüksiyonu için üreticinin talimatlarına göre bir Dronpa-Bağlayıcı-Dronpa ihtiva eden bir yapı Ramos B lenfositleri transdüksiyonu. Bir ön göre Hasat ve sayım hücreleribir önce protokolü 13 yayınladı. </li><li> Bir konsantrasyonda yeniden süspanse hücreler 1 x 10 6 orta / ml (RPMI 1640,% 10 ısı ile inaktive edilmiş FCS, 2 mM L-glutamin, 100 U / ml penisilin, 100 U / ml streptomisin, 50 uM 2-merkaptoetanol) ve bir cam tüp FACS aktarabilirsiniz. </li><li> Bir akış sitometresinde içine tüp takın ve Dronpa (GFP kanal) 14,15 floresan yoğunluğu ölçmek. </li><li> muhtemelen zararlı dalga boylarında gözleri korumak için diğer tüm adımlar için koruyucu gözlük kullanın. </li><li> El ile borunun dış 500 nm LED ışık yerleştirin ve Dronpa floresans yoğunluğunu ölçülmesi devam edin. </li><li> Dronpa floresan yoğunluğu azalır kadar LED ışıklar sayısı ve / veya yoğunluğunu arttırmak. </li><li> El ile borunun dış 400 nm LED ışık yerleştirin ve Dronpa floresans yoğunluğunu ölçülmesi devam edin. </li><li> Dronpa fluo kadar LED ışıklar sayısı ve / veya yoğunluğunu arttırmakyoğunluk artar rescence. </li><li> adımlar 1.1.5 ve 1.1.7 de photoswitching gerekli olduğu tahmin olarak cihazı oluşturmak için en az sayıda LED ışıkları kullanın. </li></ol></li><li> cihazı bina NOT: Cihaz bir profesyonel işleme dükkanı, Freiburg Üniversitesi&#39;nden Arbeitsgruppe Technik tarafından yaptırılmıştır. Çoğu parça özel yapılmış ve piyasada mevcut değildir. Her bir parçanın tam boyutları, Şekil 1 ve 2 &#39;de gösterilmiştir. Bu cihazın düzeneğinin açıklamak için, Ek Video 1 sağlanır ve en önemli adımlar aşağıda tarif edilmiştir. <ol><li> Şekil 1A gösterilen kesin ölçümler ile polivinil klorür (PVC) Mill parça A. </li><li> Parça A delinmiş deliklere LED ışıkları yerleştirin ve su sızıntısını önlemek için PVC yapıştırıcı ile her delik mühür. </li><li> Bir çok kanallı trafosuna LED&#39;leri bağlayın (her dalga boyu ayrı Chann işgal etmelidir0-29 mA ayarlanabilir çıkış gücüne sahip el). </li><li> fiziksel hasardan LED korumak için gösterildiği gibi parça A 3 vida ile dış kılıfı (B) takın. </li><li> Parça A delinmiş deliklere tüp klipleri (C) bir su pompası, tutkal cihazı bağlamak mümkün (Şekil 1a tasvir, kesit Y: 9 mm ve 61.5 mm) PVC yapıştırıcı kullanarak. </li><li> Bir 58.5 mm uzunluğunda pleksiglas tüp (D1) kesin. </li><li> Şekil 2&#39;de tasvir edildiği gibi Pleksiglastan adet D2 ve D3 imalatı. </li><li> Pleksiglas yapıştırıcı ile D1 altına Tutkal parçası D3. </li><li> parça D2 bir lastik O-ring takın ve pleksiglas yapıştırıcı ile D1 üstüne tutkal. NOT: adet D1, D2 ve D3 birlikte parça D formu </li><li> 3 vida ile parça A&#39;ya parça D yerleştirin. </li><li> transformatör güç uygulamadan önce su kaçağı cihazı test edin. </li></ol></li></ol> 2. Bir optogenetic Aracı Kinetiği Ölçme <ol&gt; <li> hücreleri hazırlayın <ol><li> RPMI-aracı madde içinde kültür Ramos B hücreleri (RPMI 1640,% 10 ısı ile inaktive edilmiş FCS, 2 mM L-glutamin, 100 U / ml penisilin, 100 U / ml streptomisin, 50 uM 2-merkaptoetanol) 3- yoğunluğunda 10 x 10 6 hücre / ml 37 ° C de ve% 5 CO2. </li><li> 350 xg 5 dakika boyunca 4 ° C&#39;de santrifüj ile hasat Ramos B hücreleri. </li><li> Üreticinin talimatlarına göre bir Neubauer sayım odası kullanılarak hücrelerin sayısı ve 600 uL ortamında 1-5 x 10 6 hücreleri tekrar süspansiyon. </li><li> Bir cam FACS tüpüne hücreleri aktarmak ölçüme kadar ışıktan buz üzerine koydu ve onları korumak. </li></ol></li><li> Flowsitometri ve cihazı hazırlayın. <ol><li> ısıtılmış su pompasına aygıtı bağlayın ve 37 ° C sıcaklığını ayarlamak. </li><li> Cam FACS tüpler kullanırken, sitometresinde bir lastik O-ring ile akış siyah O-ring değişimi ve buna ek olarak silikon gr uygulamakkolaylaştırmak. </li></ol></li><li> Ölçüm <ol><li> Dikkatle cihazın içine cam FACS tüp takın ve (Şekil 3) akış sitometresi bağlayın. </li><li> ölçümünü başlatmak ve Dronpa floresan yoğunluğu (GFP kanalı) kaydedin. </li><li> sırasıyla 400 nm veya 500 nm ışık ile hücre örneği aydınlatın ve Dronpa floresan (GFP kanalı) kaydedin. </li></ol></li><li> Veri analizi <ol><li> Yazılımın sitometresi uygun akış ile deneysel verileri analiz edin. yana dağılım ve kapısı canlı hücreler karşı komplo ileri dağılım. zamanla Dronpa floresan çizilir. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

LED Thermo Akış bir akış sitometresinin içinde optogenetic araçları incelemek için yenilikçi bir cihazdır. Şimdiye kadar, optogenetic numuneler sadece mikroskopi lazerler veya el feneri cihazlarla 11,12 ile aydınlatılan edilmiştir. örnek el feneri açısı ve mesafeye bağlı olarak, aydınlatma miktarında önemli değişkenlik deneyler arasında bekleniyor. Ayrıca, tek bir kişi bir deney çalışabilir fenerleri sayısında bir sınır yoktur. Bu deneysel repert...

Onlar yolları 1-4 sinyal kablolarını deşifre etmek için kullanılabilir, çünkü optogenetic araçlar kısmen, popülerlik kazanmaktadır. Işıkla aydınlatıldığında, kendi yapısını ve bağlanma afinitesini değiştirme fotoaktıfleştırılebılır proteinlerin yeteneğine dayanmaktadır. Sinyalizasyon elemanları bu proteinlerin kaynaştırarak karmaşık hücre içi sinyal yollarının 5-12 içinde tek bir oyuncunun belirli düzenlenmesi için izin verir. Sonuç olarak, bir sinyal yolu yüksek zamansal ve mekansal çözünürlük ile ele alınabilir. Çoğu hücre bazlı Optogenetic çalışmalarda biyokimyasal analiz 11,12 ardından ışık varlığında kültürlenmesi ile birlikte mikroskopi dayalı yöntemler kullanmaktadır. Bunun aksine, akış sitometresi kılcal boyunca hücrelerin singularizes ve hücre boyutu, boyutu ve floresan yoğunlukları ölçülür. Bu yöntem thousan analiz yeteneği dahil olmak üzere mikroskopi veya biyokimyasal yöntemlerle üzerinde önemli avantajlara sahiptirÇok kısa bir süre içinde tek hücreli çözünürlükte yaşayan hücrelerin DS. Bu nedenle, akış sitometrisi ile optogenetics birleştirilmesi arzu edilir. Bildiğimiz kadarıyla, optogenetic flow sitometri için kurulan protokol yoktur. Bir yaygın olarak kabul prosedür el feneri cihazları ile reaksiyon tüpü dışından hücreleri aydınlatmaktır. Bununla birlikte, akış kılavuzu aydınlatma sitometresi canlı hücre görüntüleme, bir silindirik, ısıtılmış su odası, reaksiyon borusu içinden geçen ve ışık gerektirir. Bu önemli bir ışık saçılması ve ışık kaybına neden olur. Ayrıca, manuel aydınlatma tarafından sağlanan ışık yoğunluğu (vb açı, mesafe,) deneyler arasında tekrarlanabilir değildir ve bir deneyde dalga boylarının sayısı için pratik bir sınır yoktur. LED Thermo Akış cihazı inşa ederek, bu sınırlamaları aşmak için başardık. Bu cihaz sayesinde, hücreler, bir sıcaklığı belirli dalga boyları ile aydınlatılabilirkısa süreli olarak kontrol edilen akış sitometrik ölçümleri sırasında bir şekilde. Bu mesafede olan ve deneyler arasında ışık hassas ve tekrarlanabilir miktarda sağlar. In vivo olarak, bizim cihazın kullanılmasını göstermek için, photoswitching sırasında Ramos B hücrelerinde Dronpa floresan sinyali kaydedilmiştir. Ramos B hücreleri, bir insan Burkitt lenfoma türetilir. Dronpa bir monomer, dimer ya da tetramer olarak var olan bir fluoresan proteindir. monomerik formda, bu ışıltısızdır. 400 nm ışık ile aydınlatma dimerizasyonu ve tetramerizasyonu uyarır ve Dronpa protein floresan vermektedir. Bu süreç, 500 nm ışık ile aydınlatma ile ters olabilir. Dronpa protein proteinler 4,13 sinyal fonksiyonu ve yerini kontrol etmek için kullanılmaktadır. Burada, bir akış sitometresinde Dronpa arasında photoswitching çalışma Ramos B hücrelerinde Dronpa-Bağlayıcı-Dronpa proteini ifade etmiştir. Bizim cihaz kullanarak, başardık verimli vegerçek zamanlı olarak floresan yoğunluğu kayıt sırasında tekrarlanabilir Dronpa photoswitch. Bu yöntem, manuel aydınlatma ile mevcut aydınlatma protokolleri üzerinden önemli avantajlar sağlamaktadır ve önemli ölçüde optogenetic araçları ve kafes bileşikleri için deneysel repertuarı genişletir. önemli ölçüde basitleştirmek ve yeni Optogenetic araçlarının keşif ve gelişimini hızlandıracak bizim aygıtının kullanılması.

<table border="0" cellpadding="0" cellspacing="0" width="1212">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass FACS tube</td>
			<td style="width:357px;">&#160;Thermo Fisher Scientific; Waltham, USA</td>
			<td style="width:312px;">14-961-26</td>
			<td style="width:296px;">Borosilicate glass tubes 12x75 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Flow Cytometer</td>
			<td style="width:357px;">BD Bioscienceg; Heidelberg, Germany</td>
			<td style="width:312px;">Fortessa II</td>
			<td>Special Order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dronpa: pcDNA3-mNeptune2-N</td>
			<td style="width:357px;">Addgene; Cambridge, USA</td>
			<td style="width:312px;">41645</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PolyJet</td>
			<td style="width:357px;">SignaGen, Rockville, USA</td>
			<td style="width:312px;">SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LED 505 nm</td>
			<td style="width:357px;">Avago Technologies; Boeblingen, Germany</td>
			<td style="width:312px;">HLMP-CE34-Y1CDD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LED 400 nm</td>
			<td style="width:357px;">Avago Technologies; Boeblingen, Germany</td>
			<td style="width:312px;">UV5TZ-400-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas tube 15 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">76999</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 3 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 2,5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 1,5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC tile 5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC tile 6 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC block 50 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RPMI</td>
			<td style="width:357px;">Invitrogen, Life Technologies; Darmstadt, Germany</td>
			<td style="width:312px;">61870-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-Mercaptoethanol</td>
			<td style="width:357px;">EMD; Germany</td>
			<td style="width:312px;">805740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">FCS</td>
			<td style="width:357px;">PAN Biotech; Aidenbach, Germany</td>
			<td style="width:312px;">P30-3302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Penicillin/Strptomycin (10000 U/ml)</td>
			<td style="width:357px;">Invitrogen, Life Technologies; Darmstadt, Germany</td>
			<td style="width:312px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acrifix plexiglas glue</td>
			<td style="width:357px;">Evonic industries, Essen, Germany</td>
			<td style="width:312px;">1R0192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tangit PVC-U glue</td>
			<td style="width:357px;">Henkel, D&#252;sseldorf, Germany</td>
			<td style="width:312px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

led thermo flow &#8212; combining optogenetics with flow cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Bir Akış Sitometre LED Thermo Flow kullanarak Cihazın bir işlevsel çekirdek ışıkları içe bakan dairesel bir biçimde düzenlenir LED silindirik bir bölme olabilir. Bu bölme ışıklarının sıcaklığı, hem de hücre örneği kontrol edilmesi için izin veren bir su ve pompa bağlanabilir. LED&#39;ler bir transformatör bağlı ve dolayısıyla her dalga boyu için ışık yoğunluğu ayrı ayrı kontrol edilebilir...

Watch this Scientific Journal Video about LED Thermo Flow Ã¢â‚¬â€ Combining Optogenetics with Flow Cytometry at JoVE.com

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED Thermo Akışı - Akım Sitometrisi ile optogenetics birleştiren

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

led-thermo-flow-combining-optogenetics-with-flow-cytometry

Research

JoVE Journal

Biology

8.5K Görüntüleme.  Max-Planck Institute for Immunobiology und Epigenetics. Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

Video: LED Thermo Flow — Combining Optogenetics with Flow Cytometry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP FCM: Sitometrisi ile Algılandı Immunoprecipitation

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Biyoloji

Flow Cytometry Purification of Mouse Meiotic Cells

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

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

Fare Mayotik Hücreler Sitometrisi Arıtma

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

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

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

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

Metabolik Etiketleme ve Flow Sitometri ile Hücre Döngüsü İlerleme Kinetik Ölçüm

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

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Akış Sitometri tabanlı Arıtma<em&gt; S. cerevisiae</em&gt; Zigot

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

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

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

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

Cep Takip Boyalar kullanarak Hücre Bölünmesi İzleme için optimize Boyama ve Çoğalma Modelleme Yöntemleri

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

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 ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

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

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

Akım Sitometri Kullanımı Ekstrasellüler Veziküller Analizi Teknikleri

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

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

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

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

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 ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

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

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

Endoreduplication izole yumru önceki Akış Sitometresi tarafından ölçme

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

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Dictyostelium discoideum Macropinocytosis akış sitometresi tarafından yüksek üretilen iş ölçümü

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...