Bu proje, Florida Devlet Scripps ve ödül sayıları sırasıyla Ulusal Genel Tıp Bilimleri Enstitüsü ve Ulusal Çocuk Sağlığı ve İnsan Gelişimi Enstitüsü R01GM085079 ve R21HD061304 paralar kısmen desteklenmiştir. Bu Scripps Araştırma Enstitüsü yazının sayısı 20.917.

Bu protokol, iki önemli adımlar ayrılabilir: (1) gerekirse fare testis hücrelerinin bölünmeler ve Hoechst 33.342 boyama, germ hücrelerinden mayoz tüm aşamaları da dahil olmak üzere ilgili mayotik kesirler, sıralama (2) FACS izledi yuvarlak spermatid. Bir kez toplanan bu zenginleştirilmiş mayotik popülasyonları geniş bir analiz için kullanılabilir. Bu protokol bir yetişkin testis ayrışma açıklar; miktarlar gençlerin veya ek testisler için buna göre adapte edilebilir. 1. Testis Ayrılma <ol><li> 15 ml&#39;lik bir tüp içinde bir decapsulated testis. </li><li> Kollajenaz tip I, 120 U / ml içeren Gey Dengesi Tuz Çözüm 3 ml (GBSS) </li><li> 10 ul DNAz I (% 50 gliserol 1mg/ml stok solüsyonu) ekleyin ve ayırmak başlangıç ​​testis tübüller görene kadar elle kuvvetlice sallayın. </li><li> 33 ° C&#39;de 15 dakika için 120 rpm maksimum yatay çalkalayın </li><li> Oda sıcaklığında dikey olarak 1 dakika için Durusu ve supernatant atın. </li><li> 1.5 ila 1.2 arasındaki adımları tekrarlayın. </li><li> Kollajenaz tip I, 50 ul, 1mm HCl çözeltisi ve 10 ul DNAz (1mg/ml) yeniden süspanse 50mg/ml tripsin stok solüsyonu içeren GBSS 2.5 ml ekleyin ve birkaç kez tüp ters. </li><li> 33 ° C&#39;de 15 dakika için 120 rpm maksimum yatay çalkalayın </li><li> Geniş delikli plastik tek yeniden süspanse kullanarak, pipet yavaşça yukarı ve aşağı 3 min.No kümeleri için bu noktada görünür olmalıdır. </li><li> Tripsin 30, 10 DNAz I, 40 DMSO (10 mg / ml) yeniden süspanse Hoechst 33.342 ul ul ul ekleyin ve birkaç kez tüp ters. </li><li> 33 ° C&#39;de 15 dakika için 120 rpm maksimum yatay çalkalayın </li><li> 400-ul fetal buzağı serumu (FCS) ve tripsin inaktive tersini ile karıştırın. </li><li> Final boyama Hoechst 33.342 50 ul DMSO (10 mg / ml) yeniden süspanse ve DNAz (1 mg / ml) 10 ul ekleyerek yapılır. </li><li> 33 ° C&#39;de 15 dakika için 120 rpm maksimum yatay çalkalayın </li><li> Ayrışmış testis örnek daha sonra iki adet 40 mikron GBSS geçirilir 50 ml konik tüp üzerinde önceden ıslatılmış tek filtreleri, tek kullanımlık Pasteur ile birkaç kez pipetleme çözüm eklenir ve örnek hafifçe karıştırılır, propidium iyodür daha sonra 5 ul (PI) pipet. </li><li> 18-gauge iğne ile örnek bir 5 ml plastik şırınga aktarılır. İkinci örnek teslimat için 22-gauge iğne ile değiştirilir. Şırınga, buz üzerinde saklanır ve ışık FACS işlem için hazır olana kadar korunur. </li></ol> 2. FACS Kurulum ve Arıtma <ol><li> Sıralama Becton-Dickinson Aria IIU hücre sıralayıcı yapıldı. Bu nedenle açıklanan koşullar, özellikle farklı ekipman kullanılarak bir başlangıç ​​noktası olarak kullanılmalıdır. Daha önce 7,8 açıklanan koşulları kullanılan. </li><li> Aria IIU UV lazer sahip olmadığından, biz Hoescht boyama 488nm bir mavi lazer ileri ve yan dağılım tespiti için kullanılan ek olarak 405nm menekşe lazer kullanarak tespit etmek için protokol uyarladı. Ayrıca, bazı filtreler, gelişmiş dağınık arsa netliği ile sonuçlanan bazı kırmızı lazer leakiness sınırlamak amacıyla modifiye edilmiştir. </li><li> Menekşe lazer Hoescht Mavi emisyon tespiti için bir 450/40nm bir bant geçiren ve Hoescht Kırmızı emisyon için bir 585/42nm bant geçiren yapılandırıldı. 502nm bir uzun pas kırmızı floresan mavi ayırmak için kullanılmıştır. </li><li> 100 mikron meme 28.000 damla / saniye bir damla sürücü frekans ile kullanılmıştır. Örnek eşik hızı yaklaşık 4000 olayları / saniye idi. Sıcaklık kontrol seçeneği, 4 örnek ve toplama tüpleri korumak için kullanılan ° C sıralama tüm süresi. Ayrıca, örnek ajitasyon özelliği tür boyunca sedimenting örnek önlemek için 200 rpm&#39;de kullanılmıştır. </li><li> Genellikle, her nüfus için 0.5-2.0x10 6 hücreli (temsilcisi bölümüne bakınız) toplanması için izin 02:58 testisler 6 saat her tür işlendi. </li><li> Örnek şırıngadan reçete yaklaşık 750μl alikotları sıralanır. Bu arada, bu duraklamalar sırasında toplama tüpleri 4 tutuldu ° C, ışıktan korumalı ve yavaşça önce sürdürme tür karışık. </li><li> % 10 FCS ile desteklenmiş 250μl DMEM içeren bir 12x75mm cam borosilicated toplama tüpünün içine sıralanır örnekleri toplandı. </li></ol> 3. Temsilcisi Sonuçlar: Tipik bir FACS profili Şekil 1&#39;de gösterilmiştir. Mayoz bölünme önemli adımlar tespit edilir ve göstergede gösterilir. Hoechst 33.342 optimal değildir leke varsa, çok daha kompakt küresel bir profil görünür ve daha az belirgin olacaktır. Yaygın bir sorun, yeterli DNAz, hızlı hücre topaklanma neden olacaktır ekleyerek görmektedir. Zan altındaki bütün adımları ekstra DNAz eklenmesi genellikle bu sorunu çözer. Freeze inciDNAz stok (-20 ° C&#39;de saklanan) Awing bu enzim aktivitesi hızlı kaybı sonucu olarak, asgari düzeyde tutulmalıdır. Spo11 mayotik rekombinasyon hotspots 12 siteleri çift iplikli tatili yönlendirir mayotik endonükleaz Şekil 2. Tipik bir prematüre mayoz 12 çift iplikli sonu oluşan bir Spo11 boş testis profilleri, yanı sıra gösterir asenkron mayoz bu ilk dalga doğasını ortaya çıkarır, 13 gün yaşlı fare, ön puberous profili. Synaptonemal kompleks protein 3 (SCP3) ve fosforile histon H2AX antikorlar kombinasyonunu kullanarak Standart sitogenetik analiz aşamasında özel mayoz bölünme işaretleri ilk olarak açıklanan 11 elswhere sıralaması mayotik hücrelerinin saflık ve doğa, teyit için kullanılan olmalıdır. <img src="/files/ftp_upload/2602/2602fig1.jpg" alt="Şekil 1" /> Şekil 1 Wild-tip mayoz FACS profilleri. A) Temsilcisi scatter plot gösterilmiştir. Kapılı hücreler gösterilmiştir. Çoğunlukla boş membranlar ve spermatid kuyrukları içeren erişkin testis bulunan enkaz büyük miktarda unutmayın. B) bir temsilci PI arsa çok sınırlı bir örnek PI pozitif hücre miktarını gösterir. Tipik bir kapı gösterilir. C) bir temsilci yabani tip Hoechst 33.342 FACS Profil gösterilir. Daha fazla çalışma için bu yöntemi kullanarak saflaştırılmış olabilir çeşitli mayotik hücre popülasyonlarının belirtilmiştir: spermatogonium (Sp), ön-leptoten (pL), leptoten zigoten (L / Z), erken Pachytene (EP), orta Pachytene ( mP), geç-Pachytene (LP), diploten (D) ve yuvarlak spermatid (SC). <img src="/files/ftp_upload/2602/2602fig2.jpg" alt="Şekil 2" /> Şekil 2 Spo11 boş ve yabani tip gün 13 mayotik FACS profilleri. A) Tipik bir Spo11 boş profil zigoten / leptoten erken pachytene geçmişte hiçbir aşamada tespit edilebilir bir açık mayotik yetmezliği ile gösterilir. B) 13 gün yabani tip erkek fare profili leptoten / zigoten hücreleri yüksek konsantrasyonda gösterir. Mayoz Bu ilk dalga olarak açık bir şekilde bu yöntemi kullanarak görüntülendi yerine, asenkron.

<ol>
	<li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Grabske, R. J., Lake, S., Gledhill, B. L., Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugal+elutriation:+separation+of+spermatogenic+cells+on+the+basis+of+sedimentation+velocity.">Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.</a> J. Cell. Physiol. 86, 177-189 (1975).</li><li>Purification, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=culture,+and+fractionation+of+spermatogenic+cells.">culture, and fractionation of spermatogenic cells.</a> Methods Enzymol. 225, 84-113 (1993).</li><li>Romrell, L. J., Bellve, A. R., Fawcett, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.+A+morphological+characterization.">Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.</a> Dev. Biol. 49, 119-131 (1976).</li><li>Meistrich, M. L., Trostle, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+testis+cells+by+equilibrium+density+centrifugation+in+renografin+gradients.">Separation of mouse testis cells by equilibrium density centrifugation in renografin gradients.</a> Exp. Cell Res. 92, 231-244 (1975).</li><li>Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr. Biol. 16, 660-667 (2006).</li><li>Lassalle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+Population'+cells+in+adult+mouse+testis+express+Bcrp1+gene+and+are+enriched+in+spermatogonia+and+germinal+stem+cells.">Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells.</a> Development. 131, 479-487 (2004).</li><li>Bastos, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis.">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65, 40-49 (2005).</li><li>Chowdhury, R., Bois, P. R., Feingold, E., Sherman, S. L., Cheung, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+variation+in+human+meiotic+recombination.">Genetic analysis of variation in human meiotic recombination.</a> PLoS Genet. 5, e1000648-e1000648 (2009).</li><li>Roig, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+TRIP13/PCH2+is+required+for+recombination+and+normal+higher-order+chromosome+structure+during+meiosis.">Mouse TRIP13/PCH2 is required for recombination and normal higher-order chromosome structure during meiosis.</a> PLoS Genet. 6, (2010).</li><li>Getun, I. V., Wu, Z. K., Khalil, A. M., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+occupancy+landscape+and+dynamics+at+mouse+recombination+hotspots.">Nucleosome occupancy landscape and dynamics at mouse recombination hotspots.</a> EMBO Rep. 11, 555-560 (2010).</li><li>Romanienko, P. J., Camerini-Otero, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Spo11+gene+is+required+for+meiotic+chromosome+synapsis.">The mouse Spo11 gene is required for meiotic chromosome synapsis.</a> Mol. Cell. 6, 975-987 (2000).</li></ol>

Burada sunulan protokol biri eş zamanlı olarak araştırmacılar, bu temel sürecin dinamiklerini incelemek için izin yetişkin erkek fareler, çok yüksek saflıkta mayotik sahne hücrelerinin tüm aralığı arındırmak için izin verir. Saf hücrelerin RNA ekstraksiyon 9,10, nucleosome haritalama 11, rekombinant molekül algılama, protein analizi, ve daha birçok arasında değişen çok çeşitli uygulamalar için kullanılabilir. Ancak, algılama yöntemleri, saflaştırılmış mayotik h...

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		<th>Material Name</th>
		<th>Type</th>
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<tr>
<td>Collagenase Type-1</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>CLSS1</td>
<td>Dissolve to 12,000U/ml in GBSS, store at 4&deg;C</td>
</tr>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>TRL3</td>
<td>Dissolve in 1mM HCl to 50mg/ml, store at 4&deg;C</td>
</tr>
<tr>
<td>Hoechst 33342</td>
<td>&nbsp;</td>
<td>Arcos</td>
<td>230001000</td>
<td>Saturated solution (10mg/ml in DMSO, store at 4&deg;C</td>
</tr>
<tr>
<td>DNAse I</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>DNEP</td>
<td>Dissolve in water/50% glycerol to 1mg/ml, store at -20&deg;C</td>
</tr>
<tr>
<td>Gey&#x2019;s Balance Salt Solution </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G9779</td>
<td>&nbsp;</td>
<tr>
<td>6" Transfer Pipet</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>137119D</td>
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flow cytometry purification of mouse meiotic cells

Testis hücre tipleri heterojen doğası ve mayoz hücre kültürü modelleri yokluğunda mayoz bölünme sırasında ortaya eşsiz bir farklılaşma programları çalışma için önemli engellerin edilmiştir. BSA ve / veya Percoll gradyanlar Yıkayarak tasviye veya Staput (sedimantasyon): İki ana yöntemleri, değişen derecelerde, hem yetişkin ve immatür hayvanlarda çeşitli mayotik kesirler arındırmak için geliştirilmiştir var. Bu yöntemlerin her ikisi mayotik hücreleri 1-5 ayrı hücre boyutu ve yoğunluğu güveniyor. Genel olarak, birkaç hücre popülasyonlarının 6 dışında, bu protokolleri detaylı moleküler analizler için gerekli olan çok sayıda mayotik hücre popülasyonlarının yeterli saflıkta verim başarısız olur. Ayrıca, bu tür yöntemleri ile genellikle tek tip mayotik hücreleri mayoz hücre örnekleri karşılaştırarak tekrarlanabilirlik ve homojenlik ile ilgili ekstra bir karmaşıklık düzeyi ekler belirli bir zaman, arındırılmalıdır. Burada, Hoechst 33.342 boyama 7,8 ile birlikte FACS kullanarak, germ hücreleri, yuvarlak spermatid, zarif bir yöntem tarif biri kolayca görselleştirmek belirlemek ve mayotik hücreleri arındırmak sağlar . Bu yöntem tüm mayotik sürecinin genel bir anlık sağlar ve bir mayoz en aşamasından son derece canlı hücreler arındırmak için izin verir. Bu saflaştırılmış hücreler daha sonra 11 mayotik rekombinasyon noktalarından örnek gen ekspresyonu 9,10 değişiklikleri ve nucleosome doluluk dinamikleri, mayoz bölünme yoluyla ilerlemesi eşlik moleküler değişiklikler için ayrıntılı olarak analiz edilebilir.

Watch this Scientific Journal Video about Flow Cytometry Purification of Mouse Meiotic Cells at JoVE.com

Flow Cytometry Purification of Mouse Meiotic Cells

Floresan aktif hücre sıralama (FACS) ile rafine bir hücre disosiasyon protokolü birleştirir fare testis yüksek derecede saflaştırılmış yaşayabilir mayotik kesirler elde etmek için etkili bir yöntem açıklanmıştır. Bu yöntem, DNA içeriği ve ayrık mayotik kesirler nükleer yoğunluğu farklılıkları yararlanır.

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 ...

flow-cytometry-purification-of-mouse-meiotic-cells

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Meiosis

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17.5K Görüntüleme.  The Scripps Research Institute. Floresan aktif hücre sıralama (FACS) ile rafine bir hücre disosiasyon protokolü birleştirir fare testis yüksek derecede saflaştırılmış yaşayabilir mayotik kesirler elde etmek için etkili bir yöntem açıklanmıştır. Bu yöntem, DNA içeriği ve ayrık mayotik kesirler nükleer yoğunluğu farklılıkları yararlanır.

Video: Flow Cytometry Purification of Mouse Meiotic Cells

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Biyoloji

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 ...

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.

LED Thermo Flow &amp;#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 ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Fare Spermatocytes segregasyonun kromozom hazırlanması yayılır

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

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 ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-iç hücre embriyonik kök hücrelerin kitle enjeksiyon daha yüksek Chimerism oranları için yol açar.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

Mikro Disseke Fare Koroid Pleksuslarından İmmün Hücrelerin İzolasyonu ve Karakterizasyonu

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vasküler Endotel Hücrelerinin Aşağı Akış Uygulamaları için Farklı Yağ Depolarından İzolasyonu ve Tanımlanması

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...