Yazarlar Epifloresans mikroskobu ile ilgili teknik danışmanlık için Dr. Leonid Volkov ve Éric Bouchard teşekkür etmek istiyorum. Finansal destek GB Sağlık Araştırması (hibe # MOP-93.781) Kanada Enstitüleri tarafından finanse

Hayvan bakım Université de Sherbrooke hayvan bakımı ve kullanımı komitesi ile uyumlu idi. 1. Tüp Hazırlığı <ol><li> Hücre sıralama bir gün önce, 5 ml&#39;lik bir polipropilen yuvarlak tabanlı tüpe ısı ile inaktive edilmiş fetal büyükbaş hayvan serumu (FBS) ile 1-2 ml ekleyin ve 15 ml ve 50 ml polipropilen konik tüplere. Kritik adım: protokolünde kullanılan her tüp kaplı olduğundan emin olun. Not: FBS kaplama boru duvarlarına yapışmasını germ hücrelerine önler. </li><li> Yavaş yavaş tüpler aynı şekilde bir rotator ile kaplamak için, 4 ° C&#39;de ters çevirme ile O / N karıştırın. </li><li> Ertesi gün, boşaltılarak kaplanmış tüpler FBS çıkarın. <ol><li> * Kritik adım: 5 ml polipropilen yuvarlak dipli tüpler ve hücre hazırlanması için kullanılan 15 ve 50 ml tüpler (protokol 2.11 2.1 adımları) tamamen havalandırmak (yan akımlarının, yani belirsiz sapma) önlemek için FBS boşalttı emin olun. FBS kaldırmak için bir P200 pipetlemeyin kullanın. <br/&gt; Not: önce hücrelerle veya hücre ayırma sırasında kullanılan tüpler kontaminasyon kaybına neden olabilir sıralama için tüplere Kalıntı FBS, </li><li> Yuvarlak 5 ml polipropilen saflaştırılmış hücreleri toplamak için kullanılan alt tüpler içinde FBS (yaklaşık 100-200 ul) küçük bir kalıntı hacmi bırakın. </li></ol></li></ol> 2. Hücre Hazırlanması <ol><li> O CO 2 boğulma ardından 2 / izofluran (daha sonra% 2 oranında muhafaza% 5 indüksiyon) ile anestezi altında tek erkek fare Kurban. </li><li> Tüketim ve açarlar hem testis ve 1.5 ml tüp içine tampon sıralama 500 ul küçük bir makas ile kıyma. </li><li> Sağlam bir 100-1,000 ul ucu ile daha sonra seminifer ilk kesik 100-1,000 ul ucu ile 1.5 ml tüp nazik yukarı ve aşağı pipetleme tübüllerinde, ve gelen yıkayın germ hücreleri. </li><li> 40 mikron hücre süzgeç kullanılarak bir filtrasyon adım enkaz ve kümeleri çıkarın ve 50 ml konik tüp prev içine süzülen toplamakiously Aşama 1 FBS kaplanmıştır. </li><li> Daha önce 1. adımda kaplı 15 ml konik tüp içine toplam 3 ml hacminde ve transfer filtrata kadar tampon sıralama ile bir kez filtreyi yıkayın. </li><li> 0,8 mM EDTA nihai bir konsantrasyon elde etmek üzere, 50 mM EDTA hücre süspansiyonu mililitre başına pH 8 (3 ml 48 ul) 16 ul ekle. </li><li> Germ hücreleri düzeltmek için, yavaş yavaş süt süspansiyon üretecek hafif ajitasyon ile 10 mi pipeti (düşük hızda vorteks) kullanılarak buz-soğuğu% 100 etanol içinde 3 hacim ekleyin. Kritik adım: yavaşça etanol ilave hücre hazırlama bütünlüğünü korumak için çok önemli ve çok önemlidir. Biz etanol hızlı ekleme etkinliğini sıralama önemli bir düşüşe neden hücre lizizine neden olduğunu kaydetti. 3 ml hücre süspansiyonu için, etanol, 9 ml, yaklaşık 1 dakika içinde ilave edilmelidir. </li><li> Ters çevirme ile ara sıra karıştırma ile 15 dakika süreyle buz üzerinde inkübe hücreleri. </li><li> Santrifüj hücre 5 dakika boyunca 800 xg&#39;de süspansiyon ve net çıkarmaksüpernatant. </li><li> Yavaşça tampon sıralama 2 ml sabit germ hücreleri tekrar süspansiyon. </li><li> SYTO16 DNA boyası (DMSO içinde 1 mM çözelti) 4 ul ekleyin ve (ışıktan korunarak), en az 30 dakika inkübe edilir. Not: iyi sıralama sonuçları kolayca spermatidlerin yüksek oranda sıkıştırılmış çekirdekler dahil edilen geçirgen bir DNA boya olduğu SYTO16 kullanılarak elde edilir. </li></ol> 3. Hücre Ayırıcı Kurma Not: Burada, bir 4-Lazer (405 nm - menekşe, 488 nm - mavi, 561 nm - sarı-yeşil, 633 nm - kırmızı) 20-parametre BDFACSAria III hücre sıralayıcı kullanılır. BD FACSDiva 6.1.3 yazılımı veri görselleştirmek ve analiz etmek için kullanılır. Ayarları kullanılır sıralayıcı türüne bağlı olarak değişebilir. <ol><li> Fluidik kapatma prosedürü (yazılımında &quot;Sitometre&quot; menüsü) sıralama öncesinde sırasında kılıf sıvısı olarak% 70 etanol çalıştırarak FACS sıralayıcı sterilize edin. Yazılımın araç çubuğundaki &quot;Sitometreyi&quot; seçin ve &quot;Fluidic sh seçin. &quot;utdown süreçler tarafından belirtilen adımları izleyin. </li><li> Hazırlama ve herhangi bir kristal ve kirletici maddelerin bertaraf edecek şekilde bir 0.22 um filtre ile 1x PBS filtre. Tankın iç üst kaynak hattına kılıf deposunu doldurunuz. </li><li> Yazılımını başlatın ve oturum açın. Önce sıralama için en az 30 dakika süreyle lazer ısınmak için FACS sıralayıcı başlatın. Akıskanlar tekni˘gi gelen etanol dışarı floş iki akışkan başlangıç ​​süreçlerini çalıştırın. Yazılımın araç çubuğundaki &quot;Sitometreyi&quot; seçin ve &quot;Fluidic başlangıç&quot; ı seçin. Süreçlerle belirtilen adımları izleyin. Not: Bu normal bir kılıf sıvısı (1x PBS) ile Fluidics geri yükler. </li><li> Sıralama blok ve distile su ile saptırma plakaları temizleyin ve iyice kurulayın. Bir sonikasyon banyosu içinde 2 dakika için damıtılmış su içinde bir boru içine yerleştirilmiş bir 100 um ağızlık sonikasyon. Iyice memeyi silin. </li><li> Akış hücre içine 100 mikron memesini takın ve 12 meme-kilitleme kolunu saat yönünde çevirinhr pozisyonu. 20 psi kılıf basıncını ayarlayın. </li><li> Dere açın ve (30 civarında) frekans hedef değerleri elde etmek için genlik ayarlayın 1 (150 civarında) bırakın ve (12 civarında) Gap ve istikrarlı bir damlacık kırılması deseni (birkaç uydu damla) kurmak. Zayıflama kapatın. </li><li> Dere düz ve sıralama bloğunun açısını değiştirerek atık aspiratör merkezini vurur emin olun. Not: 10 psi A 130 mikron meme de test edildi ve belirgin bir fark bulunmuştur. </li><li> Sarı-yeşil lazerler için, mavi mor kırmızı için -77.39, 37.33 -39.85 ve için 0 olarak ayarlayın lazer gecikme. </li><li> Sarı-yeşil lazerler için, mavi mor kırmızı için 1.0, 0.75 ve 0.96 için 1.14 ölçekleme ayarlayın alanları. </li><li> (Yazılım &quot;Yan Akım&quot; penceresinde) Test Sıralama kullanarak, yan akışları havalandırarak olmadığından emin olun. Onlar middl isabet böylece &quot;Yan Akım&quot; penceresinde, her yan akımının gerilim sürgü ayarlamakTest toplama tüpünün e. Merkez akışı sıkı değilse, merkez akışı sınırlamak için 2 nd, 3 rd ve 4. damla ayarlarını. </li><li> Yazılımın (&quot;Sitometre&quot; menüsü) Sitometrenin Kurulum ve İzleme (CS &amp; T) uygulamasını başlatın. </li><li> Üreticinin talimatlarını kullanarak sitometresinin performans karakterizasyonu ve izleme otomatikleştirmek için 350 ul başına 1 damla sitometresinin kurulum ve izleme boncuk kullanın. </li><li> CS &amp; T uygulaması çıkın. CS &amp; T ayarı uygulamak, dere parametreleri hala istikrarlı bir damlacık kırılması modelini izin verdiğinizden emin olun, ve (&quot;kırılması penceresinde&quot; olarak) üreticinin talimatlarına açıklandığı gibi Sweet Spot butonuna açın. </li><li> Floresan boncuklar kullanarak Bırak Gecikme değerini optimize (Malzeme Tablo). <ol><li> Tutucuya toplama tüpleri yükleyin. Sıralama bloğu kapağını açın. &quot;Yan Akım&quot; penceresinde, &quot;seçeneğini ardından gerilime açınTest sıralama &quot;ve çekmeceyi açın ve toplama tüpleri erişimi için Aspiratör Çekmece düğmesini tıklatın. </li><li> Onlar tüp merkezine gitmek böylece yan akışları optimize edin. Gerektiği gibi değer ve kaydırma çubuklarını ayarlayın. </li><li> Sıralama bloğu kapağını kapatın ve merkezde parlak boncuk nokta elde etmek mikrometre kadranını ayarlamak için emin olun. Unselect atık çekmece test sıralama ve gerilim. Toplama tüpleri çıkarın. </li><li> Üreticinin talimatlarına göre açılan gecikme optimize edin. <ol><li> Kısaca, boncuk şişe kuvvetlice sallayın ve PBS 0.5 ml boncuk 1 damla sulandırmak. </li><li> Üreticinin talimatlarına kullanarak, yazılım mevcuttur Bırak Gecikme deneme şablonu kullanarak damla gecikme ayarlayabilirsiniz. Alternatif olarak, açılan gecikmesini ayarlamak için Otomatik Bırak Gecikme özelliğini kullanın. </li><li> Floresan boncuk tüp yerleştirin ve eşik oranı kadar ~ 3.000 4.000 olaylar / saniye akış hızını ayarlayın. Sıralama hassasiyetini ayarlamak için &quot;İlk&quot; ve sonly &quot;Sıralama düzeni&quot; penceresindeki &quot;İnce ayar&quot; için. Optik filtreyi seçin ve boncuk sıralayın. % 100 sola sıralanır ~ kadar Bırak Gecikmesi ayarlayın. Not: Bu adım emin olmak için kritik olduğunu bir yan akışına faiz tür hücreler. Boncuk ve optik filtre, bir% 100 damlacık sapma yakın elde etmek için kullanılır. </li></ol></li></ol></li><li> FSC dedektörü bloğunun sol ucundaki bir FSC 1.5 ND (nötr yoğunluk) filtreyi yerleştirin. 2.00 us pencere uzantısı ve (&quot;Sitometre&quot; penceresindeki &quot;Lazer&quot; sekmesinde) 1.00 FSC Alan ölçekleme ayarlayın. </li><li> Örnek tüpü yerleştirmeden önce, topaklanma önlemek için örnek hattının sonunda 50 um örnek filtre satırı koyun. Bunu yapmak için, &quot;Sitometre&quot; menüsünden &quot;Örnek Filtre değiştirme&quot; seçeneğini seçin. </li><li> Yan akım arasındaki havalandırmadan iyi bir şekilde ayrılmasını sağlamak ve Polipropilen yapışmasını önlemek için hücrelerin örnek sıvı bileşimin testne tüpler. <ol><li> , Örnek tüp Yük saptırma plakaları açın ve çekmece kapalı &quot;Test tür&quot; seçeneğini seçin. Ayrılık çok fazla ventilatör olmadan oluşursa yan dereler bak. Not: Havalandırma, numune tamponu içinde, yüksek protein konsantrasyonunda muhtemeldir. </li></ol></li><li> FACS aracı haline örnek tüp yükleyin. 300 rpm&#39;lik bir ajitasyon örneği ve 4 ° C&#39;de bir örnek sıcaklığı seçin. &quot;Acquisition Dashboard&quot; in &quot;Veri Edinme&quot; seçeneğini seçin. </li><li> Gerekirse, (3.0 maksimum akış hızında) / saniye 5000 etkinlikleri en fazla, uygun bir olay hızı elde etmek için örnek seyreltin. Sıralanmış hücrelerin saflığını muhafaza edecek şekilde Strictly bu sınırları saygı. </li><li> Analiz ve hücreleri sıralamak için, faiz nüfusu müdahale etmeden enkaz ortadan kaldırmak için FSC eşik değerini optimize. Logaritmik ölçekte yapmak amacıyla 530/30 filtresi ile foto-çoğaltıcı tüp (PMT) dedektörün voltajını ayarlamakEmin toplam nüfusun floresan sinyal ölçeğinde dahilinde ve pozitif nüfus kapısı (Şekil 1, Kapı 1). </li><li> Bir Forward Scatter-alanı (FSC-A) / Side Scatter alan SYTO16 pozitif nüfusun (SSC-A) arsa üretin ve yaklaşık 110 V Doğrusal ölçekte için FSC-A ayarlayın ve yaklaşık 150 V SSC-A Çok büyük hücreleri ve hücre agrega hariç ise logaritmik ölçekte tüm olayları görselleştirmek için. </li><li> &quot;Sıralama Düzeni&quot; penceresinde, &quot;Sıralama Hassas Mode&quot; set &quot;4-yollu saflık&quot; için (Verim maskesi: 0, Saflık Maskesi: 32, Faz Maskesi: 0). Sürekli sıralama için &quot;Hedef Etkinlikler&quot; menüsünden &quot;Sürekli&quot; yi seçin. </li><li> (&quot;Sıralama Düzeni&quot; penceresinde) tüplere karşılık gelen alanda sıralamak için popülasyonları ekleyin. Yüksek orta frekans ve ucunda düşük frekanslarda olanlar ile yerleştirin popülasyonları. , Sıralama sırasında 2500 V gerilim plakaları ayarlayın örnek toplama t tutmakBuz üzerinde UBE. </li></ol> 4. Hücre Ayırma <ol><li> Hemen sıralamadan önce, yuvarlak dipli tüp önceden kaplanmış 5 ml polipropilen 50 mikron filtre kullanılarak filtre hücreleri. </li><li> Tampon sıralama 1-2 ml yoğun filtreyi yıkayın. </li><li> Şekil 1&#39;de detaylı yolluk planı izleyen bir 488 nm lazer donanımlı hücre sıralayıcı kullanılarak sırala sabit germ hücreleri. Daha önce buz üzerinde bölüm 1 kaplı 5 ml polipropilen yuvarlak dipli tüplerde arıtılmış kesirler toplayın. <ol><li> FBS 100-200 ul tüp duvarına sıralanmış hücrelerin yapışmasını önlemek için toplama tüpleri muhafaza olduğundan emin olun. </li><li> SYTO16 konsantrasyonu sıralama döneminde floresan dalgalanmaları önlemek için doygunluğa emin olun. Ayırma Grafiklerdeki Alexa Fluor 488 parametresi (DNA boyama) hücre boyama yoğunluğunun daha fazla değişiklik olduğunda SYTO16 satürasyon. Gerekirse, 1 ulSıralama tüp SYTO16 doygunluk ulaşmak için. </li><li> Alexa Fluor 488-A vs olay sayısı: aşağıdaki parametreleri gösteren bir grafik üzerinde toplam etkinlik gösterir. </li><li> Şekil 1 &#39;de gösterildiği gibi, Gate 1 pozitif bir DNA boyama hücreleri seçin. <ol><li> Parametre olarak FSC-A vs SSC-A kullanılarak bir grafik üzerinde kapısı 1 Ekran hücreleri <ol><li> Şekil 1&#39;de gösterdiği gibi, Gate 2 ile hücreleri seçin. </li><li> Parametre olarak Alexa Fluor 488-A vs FSC-A kullanılarak bir grafik üzerinde Gate 2 Ekran hücreleri. </li><li> Şekil 1&#39;de gösterdiği gibi, Gate 2 grafik üzerinde Gate 5 ve Gate 6 hücreleri seçin. </li><li> Parametre olarak Alexa Fluor 488-A vs 488-W Alexa Fluor kullanarak grafik üzerinde ayrı ayrı Gate 5 ve 6 Gate gösterir. </li><li> Seç Spermiyogenez Gate 5 grafikte 1-9 spermatid adımları ve Şekil 1&#39;de gösterdi Spermiyogenez Gate 6 grafik 10-12 spermatid adımları yineleyin. </li></ol></li><li> Ekranparametre olarak Alexa Fluor 488-A vs FSC-A kullanılarak bir grafik üzerinde kapısı 1 hücreleri <ol><li> Şekil 1&#39;de gösterdiği gibi, Gate 3 ve Gate 4 hücreleri seçin. </li><li> Parametre olarak Alexa Fluor 488-A vs 488-W Alexa Fluor kullanarak grafik üzerinde ayrı ayrı Gate 3 ve Gate 4 görüntüler. </li><li> Seç Spermiyogenez Kapısı 3 grafik üzerinde 13-14 spermatid adımları ve Şekil 1&#39;de gösterdi Spermiyogenez, Gate 4 grafik üzerinde 15-16 spermatid adımları yineleyin. </li></ol></li></ol></li></ol></li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trinucleotide+expansion+in+haploid+germ+cells+by+gap+repair.">Trinucleotide expansion in haploid germ cells by gap repair.</a> Nature Genet. 27 (4), 407-411 (2001).</li><li>Bastos, H., 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=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 (1), 40-49 (2005).</li><li>Lassalle, B., Ziyyat, A., Testart, J., Finaz, C., Lef&#232;vre, A. <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+method+to+isolate+round+spermatids+from+mouse+testis.">Flow cytometric method to isolate round spermatids from mouse testis.</a> Hum. Reprod. 14 (2), 388-394 (1999).</li></ol>

The authors declare no competing financial interests.

Spermatogenik hücreler her zaman seminifer epitel karmaşıklığı yanı sıra, in vitro kültür sınırlı bir başarı verilen araştırmaya zorlu oylandı. Yıllar boyunca, pek çok yaklaşım, çeşitli türlerden germ hücreleri geliştirildi saflaştırmak için. Percoll ya da sığır serum albümini gradyanları ile gravite arıtma kullanılarak sedimentasyon teknikler genellikle sağlam germinal hücrelerde daha iyi bir verim sağlar, ancak bu mayoz tetraploid hücre ve spermatidler 10 gi...

Haploid round spermatids differentiate into spermatozoa by a process called spermiogenesis. This involves many different steps including the acquisition of a flagellum, chromatin and cytoskeleton remodeling, condensation of the nucleus as well as the loss of most of the cytoplasm. These unique cellular events must be finely regulated in order to produce a mature functional gamete with an intact genome suitable for fertilization. Spermiogenesis can hardly be studied in vitro since no reliable cell culture system has so far been able to support progression through the different steps of the process. Moreover, actual in vitro techniques lead to a poor yield1,2. In vivo, proper transitions through the different steps of spermiogenesis are crucial for the natural functional integrity of the male gamete. Successful purification of spermatids according to their differentiation steps has never been accomplished with a level of enrichment sufficient to allow molecular characterization of spermiogenesis. For instance, purification of key steps of the spermatidal differentiation would be especially useful to study the developing acrosome, formation of the midpiece3, cell junction dynamics4, RNA dynamics5, chromatin remodeling process6,7 or genomic stability8. Purification of spermatids has been hampered by their progressive morphological transformation, the lack of known stage-specific external biomarkers, and their peculiar shape and size. 
Although most male germ cells display a direct relationship between DNA staining and ploidy (DNA content), we noticed that such positive correlation is no longer applicable to spermatids. This stems from our early observation that seminiferous tubule sections show variable intensity of DNA staining throughout the different spermiogenesis steps. Although DNA staining is consistent with their haploid set of chromosomes from spermiogenesis steps 1 to 7 (round spermatids), we observed a sharp increase in fluorescence intensity with DAPI or SYTO 16 around the onset of nuclear reorganization and chromatin remodeling (spermiogenesis step 8) reaching a peak at the onset of nuclear condensation (spermiogenesis steps 11-12). Following condensation of the nucleus, DNA staining intensity decreases until spermiation (spermiogenesis step 16). We surmised that this was likely associated with the formation of their peculiar chromatin structure transition where histones are replaced by protamines. We therefore developed a reliable flow cytometry method that allows the separation of spermatids using the variation of DNA intensity of spermatids as a main selection parameter.
A simple flow cytometry approach is described to separate mouse spermatids with high purity (95-100%) based on their apparent DNA content (SYTO16 staining), size and granulosity. Spermatids are separated into four populations; spermiogenesis steps 1-9, 10-12, 13-14 and 15-16. Purified spermatids are suitable for genetic/genomic analysis, as well as proteomic applications as described in a recent publication from our group9.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Isoflurane</td>
			<td style="width:119px;">ABBOT</td>
			<td style="width:129px;">05260-05</td>
			<td style="width:184px;">For mouse anesthesia before euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Fetal bovine serum</td>
			<td style="width:119px;">Wisent</td>
			<td style="width:129px;">90150</td>
			<td style="width:184px;">For tube coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">1X PBS</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">EDTA</td>
			<td style="width:119px;">BioShop</td>
			<td style="width:129px;">EDT</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">HEPES</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:129px;">H</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">100 % Ethanol</td>
			<td style="width:119px;">Les alcools de commerce</td>
			<td style="width:129px;">092-09-11N</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">SYTO 16</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:129px;">S7578</td>
			<td style="width:184px;">DNA staining</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">5 ml polypropylene round bottom tubes</td>
			<td style="width:119px;">BD Falcon</td>
			<td style="width:129px;">352063</td>
			<td style="width:184px;">Sorted cells collection</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">15 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">1500</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">50 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">5000</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">TEC4 anaesthetic vaporizer</td>
			<td style="width:119px;">Ohmeda</td>
			<td style="width:129px;">1160526</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">CO2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">C799117902</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">O2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">O254130501</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Homemade mouse gas chamber</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">40 &#181;m Falcon cell strainer</td>
			<td style="width:119px;">Corning Incorporated</td>
			<td style="width:129px;">352340</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">50-micron sample line filters</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">649049</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Vortex mixer</td>
			<td style="width:119px;">Labnet international, inc.</td>
			<td style="width:129px;">S0200</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Dynac centrifuge</td>
			<td style="width:119px;">Clay Adams</td>
			<td style="width:129px;">101</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Celltrics 50 &#181;m filters</td>
			<td style="width:119px;">Partec</td>
			<td style="width:129px;">04-004-2327</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">488 nm laser-euipped cell sorter</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">FACSAria III</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:135px;">Accudop Fluorescent Beads</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">345249</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">Sorting Buffer: 1X PBS, 1mM EDTA pH 8.0, 25mM HEPES pH 7.0, 1%FBS</td>
			<td></td>
			<td></td>
			<td style="width:184px;">FBS is heat-inactivated. Make fresh solution, 0.22 &#956;m filtered and keep at 4&#176;C.</td>
		</tr>
	</tbody>
</table>

step-specific sorting of mouse spermatids by flow cytometry

Fare spermatidler farklılaşması sağlam bir genomu olan bir fonksiyonel erkek gamet üretimi için bir kritik işlem sonraki kuşağa olmaktır. Şimdiye kadar, bu morfolojik geçiş moleküler çalışmalar daha sonraki analizler için spermatid farklılaşma bu önemli adımlardan yeterli ayrılmasını sağlayan bir yöntem olmaması nedeniyle sekteye oylandı. Akış sitometri kullanılarak bu hücrelerin düzgün gating de önceki girişimler için kromatin yenileme geçiren spermatidler DNA floresan kendine özgü bir artış zor olabilir. Bu gözleme dayanarak, biz etanol ile sabit fare spermatid dört popülasyonlarının tekrarlanabilir arıtılmasını sağlayan düzeni sitometri basit bir akış ayrıntıları, her nükleer yeniden şekillenme sürecinde farklı bir durumu temsil. Nüfus zenginleştirme aşaması özgü işaretleri ve morfolojik kriterler kullanılarak teyit edilir. Saflaştırılmış genomik spermatidler ve proteom için kullanılabiliric analizleri.

Yolluk strateji akış sitometri ile kullanılabilir Şekil 1 dört derece saf spermatid popülasyonları sıralamak için flow sitometri kullanılan yolluk stratejisini temsil eder. Kısaca, pozitif DNA boyama hücreleri (Alexa Fluor 488-A) Birinci boyut vs granulosity (SSC-A) gösteren bir nokta arsa üzerinde Spermiyogenezin 1. spermatid 1-12 seçilir adımlar Kapısı (Gate 2) ile seçilir (FSC Kapı 1. A)...

Watch this Scientific Journal Video about Step-specific Sorting of Mouse Spermatids by Flow Cytometry at JoVE.com

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

Adım özgü Fare spermatidlerin Sıralama&#39;yı Akışı Sitometrinin tarafından

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

step-specific-sorting-of-mouse-spermatids-by-flow-cytometry

Research

JoVE Journal

Developmental Biology

10.5K Görüntüleme.  Université de Sherbrooke. We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

Video: Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Akım Sitometresi ile Fare Embriyosunda Eritromiyeloid Progenitörlerin Ve Döllerinin Belirlenmesi

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Gelişim Biyolojisi

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

Yalıtımlı ve Akış Sitometrisi ile Pankreas mezenkim ve Hücreler Analizi

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Memeli Erkek Germ Hücrelerinin Çok Denekli Saflaştırılmasında Standart Bir Yaklaşım, Mekanik Doku Ayrılma ve Akış Sitometrisi ile

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Hematopoetik kök hücre transplantasyonu myelodisplastik sendromu kökeni değerlendirmek için kullanımı

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Kalsiyum akını, gözenek oluşumu ve P2X7 reseptörleri yetişkin nöral progenitör hücreler tarafından fagositoz araştırmak için gerçek zamanlı canlı hücre Akış Sitometresi

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

Kütle sitometrisi ile hücresel protein Ifadesinin hücre döngüsü spesifik analizi için Mouse cilt keratinositlerin yalıtım ve boyama

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

Standart Laboratuvar Ekipmanları Kullanılarak Fare Testlerinden Pachytene Spermatosit ve Spermatidlerin Zenginleşmesi

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

Gelişmekte Olan Zebra Balıklarında Değiştirilmiş Kan Oluşumunu Tespit Etmek ve Nicel hale Getirmek için Akış Sitometrisini Kullanma

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

FACS-İzolasyon ve Fibro-Adipojenik Progenitörlerin ve Kas Kök Hücrelerinin Bozulmamış ve Yaralı Fare İskelet Kasından Kültürü

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Patlama Oluşturan Birim ve Koloni Oluşturan Birim Eritroid Progenitörlerinin Fare Dokusundan Akım Sitometrisi ile Tanımlanması ve İzolasyonu

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...