We thank Flow Cytometry Laboratory at Virginia-Maryland College of Veterinary Medicine for the use of flow cytometry core facility. This work was supported by XML's startup funds. XL is a Stamps Fellow in the Biomedical and Veterinary Sciences graduate program.

NOT: MRL / Mp-Fas lpr lupus eğilimli fareler yetiştirilen ve Virginia Tech (Hayvan Refahı Güvencesi sayısı Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) gereklerine itibaren belirli bir patojen içermeyen tesiste muhafaza edildi (MRL / lpr): A3208-01). Bu çalışma Ulusal Sağlık Enstitüleri Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu önerileri ile tam uyum içinde yürütülmüştür. Tüm hayvan deneyleri IACUC protokolü # 12-062 altında gerçekleştirilmiştir. 1. Hücre Kültürü Orta ve Sıralama Tampon <ol><li> Tüm hücre kültürü ortamı (C 10),% 10 fetal sığır serumu, 1 mM sodyum piruvat,% 1, 100 MEM temel olmayan amino asitler, 10 mM HEPES, 55 uM 2-merkaptoetanol, 2 mM L-glutamin ile takviye edilmiş RPMI 1640 ile hazırlayın 100 U / ml penisilin-streptomisin. </li><li> ayırma tamponu hazırlayın 10 mM HEPES ile desteklenmiş 1 x Hank&#39;in dengeli tuz çözeltisi (HBSS) ile (dolu HBSS), 2.5 mg / ml sığır serumu albümini,0,05 mM MgCl2 ve 0.2 U / ml DNaz I </li></ol> 2. Fare diseksiyonu <ol><li> 4 ml C10 içeren iki 6 cm çapında yemekleri hazırlayın. buz üzerinde yemekleri yerleştirin. </li><li> CO 2 inhalasyon yoluyla fare Euthanize. </li><li> dalak hasat ve buz üzerinde C10 ile bir tabak içinde tutmak. <ol><li> diseksiyon plaka üzerinde yukarı bakacak şekilde karın fare aşağı pin. % 70 etanol püskürtülerek vücudu sterilize edin. </li><li> Cildi kesin ve altında boyun pubis eklemi ventral orta hat boyunca kas duvarından cilt ayırmak. </li><li> Ayak bileği ilk kesi arka bacak boyunca cildi kesin. </li><li> arka yüzlerine cilt pin ve diyafram ilk kesi yerden kenarlarında kas duvarı kesti. </li><li> kafanın sağ tarafında kas duvarı geri pin. </li><li> ince bağırsak altında ve mide yanında farenin sağ tarafta dalak bulun. inci bir ucunu Pick upe dalak ve mide ayırın. </li></ol></li><li> femur ve tibia da dahil olmak üzere, her iki arka bacaklarda kesip, ve mümkün olduğunca tüm kasları kaldırın. <ol><li> kan damarları önlemek için çalışıyoruz, keskin bir makas ile ayak bileği pelvik / kalça ekleminden arka bacak boyunca kasları kesti. </li><li> Kemik iliği içeriğinin maruz kalmadan pelvis / kalça eklemi ve ayak bileği / ayak eklem keserek arka uzuv ayırmak. </li><li> tekrar tekrar jilet ile çizilme ve ortak noktalarda kasları keserek kemikler üzerinde temiz kalan kaslar. </li></ol></li><li> buz üzerinde 4 ml C10 içeren 6 cm çanak kemikleri tutun. Bir sonraki fare diseksiyon geçin. </li></ol> 3. splenosit yalıtımı <ol><li> Resim kırmızı renkli görünür olana kadar 4 mi C10 ihtiva eden çanak üzerine 70 mikron hücre süzgecinden karşı şırınga pistonu yavaşça dalak homojenize edilir. </li><li> süzgeç a ile çanak içine ilave 6 ml C10 eklend sonra 15 ml konik tüp toplam 10 ml hücre süspansiyonu aktarın. </li><li> 5 dakika, 4 ° C, 800 xg&#39;de konik tüp santrifüjleyin. </li><li> Santrifüj işleminden sonra, süpernatan aspire ve 2 ml 1 x kırmızı kan hücresi (RBC) liziz tamponu içinde hücre pelletini. 5 dakika boyunca oda sıcaklığında inkübe edin. </li><li> İnkübasyondan sonra, 5 dakika, 4 ° C, 800 x g hızında santrifüj konik tüp. </li><li> Santrifüj işleminden sonra, süpernatan aspire ve 1 ml C10 hücre pelletini. herhangi bir büyük kümeleri (ölü hücreler) atın ve daha sonra buz üzerinde tüp tutun. NOT: Birçok küçük kümeleri varsa, bir kez hücre süspansiyonu filtre 70 mikron hücre süzgeç kullanın. </li><li> otomatik bir hücre sayacı tripan mavisi ile hücre sayısı. </li></ol> 4. Kemik iliği Hücre İzolasyonu <ol><li> bir havan içine 4 mi C10 kemik aktarın ve toplam 10 mi C10 lik bir hacim oluşturmak için 6 ml C10 ekleyin. </li><li> ap kullanarak harç yavaşça kemikleri çatlakestle. </li><li> C10 içine kemik iliği serbest bırakmak için tokmak ile hafifçe karıştırın. </li><li> 70 mikron hücre süzgecinden buz üzerinde bir 50 ml konik tüp içine kemik iliği içeren 10 ml C10 aktarın. hala kemikleri içeren harcın içine 10 ml taze C10 ekleyin. NOT: Pipet kırmızı kümeleri görülebilir ise süzgecinden geçirmeden önce kemik iliği yukarı ve aşağı birkaç kez içeren solüsyon. </li><li> Tekrarlayın kez 4.4&#39;e 4.2 adımları tekrarlayın. Son yıkamadan sonra, kemikler, beyaz görünmelidir. </li><li> 5 dakika, 4 ° C, 800 x g&#39;de 50 ml konik tüp santrifüjleyin. Öte yandan oda sıcaklığında bir 15 mL konik santrifüj tüpü içinde 5 ml Ready-to-use yoğunluk gradyan hazırlarlar. </li><li> Santrifüj işleminden sonra, süpernatan aspire ve 10 ml C10 hücre pelletini. </li><li> Yavaş yavaş iki faz arasındaki net arayüzü koruyarak, 5 ml yoğunluk gradyan ortamının üstüne 10 ml hücre süspansiyonu katman. Daha sonra 30 dakikada için 1.363 g 15 ml konik tüp santrifüj0 (Fren KAPALI) olarak ayarlanmış hızlanma 9 olarak set ve yavaşlama ile RT (20 ° C). </li><li> Santrifüj işleminden sonra, dikkatle üst 8 ml solüsyon kaldırmak ve yeni bir 15 ml konik tüp içine arabirimi (mononükleer hücrelerin tabakası) 2 ml buffy coat toplamak. </li><li> Kemik iliği tek çekirdekli hücreleri, kap ihtiva eden 15 ml konik tüp, 12 ml buz soğukluğunda C10 ekleyin ve daha sonra iyice karıştırın, 10 dakika, 4 ° C, 800 x g hızında santrifüj tüpü birkaç kez ters çevirin. </li></ol> FACS 5. Hücre Yüzey Boyama <ol><li> örnek boyama <ol><li> anti-fare CD16 / 32 antikor çözeltisi (İN 1-100 seyreltme HBSS-dolu / ml son konsantrasyon 5 ug) hazırlayın. </li><li> Adım 4.10 santrifüjlemeden sonra, süpernatan aspire ve tek çekirdekli hücreler üzerindeki Fc reseptörü bloke etme 100 ul anti-fare CD16 / 32 antikor çözeltisi hücre pelletini. 10 dakika boyunca buz üzerinde inkübe edin. </li><li> (Karınca floresan konjuge antikor karışım hazırlayınI-fare CD11c-PE 1:40 seyrelti, anti-fare CD11 b-APC-Cy7 1:80 seyrelti, anti-fare PUKÖ 1-FITC 1:40 seyreltme ve anti-fare B220-V500 HBSS- bölgesindeki 1:40 seyreltme örnek başına 100 ul nihai hacim) üretici tarafından tavsiye edildiği gibi, tam. NOT: Kullanmadan önce antikorlar titre önemlidir. </li><li> Aşama 5.1.2 içinde 10 dakika inkübe edildikten sonra, anti-fare CD16 / 32 bağlanmamış kaldırmak için 5 dakika, 4 ° C, 800 x g&#39;de 4 mi buzla soğutulmuş HBSS dolu ve santrifüj ekleyin. </li><li> Santrifüj işleminden sonra, süpernatan aspire ve 100 ul floresan konjuge antikor karışımı içinde hücre pelletini. karanlıkta 15 dakika süreyle buz üzerinde inkübe edin. </li><li> 15 dakika inkübasyondan sonra, bağlanmamış floresans-konjuge antikor kaldırmak için 5 dakika, 4 ° C, 800 x g&#39;de 4 mi buzla soğutulmuş HBSS dolu ve santrifüj ekleyin. </li><li> FACS yükleme solüsyonu (HBSS-tam 500 ng / ml DAPI) hazırlayın. </li><li> Aşama 5.1.6 santrifüjlemeden sonra, süpernatan aspire 500 mikron hücre pelletiniL FACS yükleme çözümü. 12 x 75 mm yuvarlak tabanlı tüpü (FACS tüpü) içine hücre süspansiyonu aktarın ve 1 saat içinde sıralama kadar karanlıkta buz üzerinde tüp tutun. </li></ol></li><li> Tazminat boyanma <ol><li> PE, APC-Cy7, FITC, V500, DAPI veya boyasız olarak etiket 6 FACS tüpleri. 1 x 10 6 hücre / FACS tüplere boru ve en kısım splenositler, 5 dakika, 4 ° C, 800 x g&#39;de 4 mi HBSS-Tam ile yıkayın. NOT: ölü hücrelerden canlı hücreleri ayırt etmek floresan boya, DAPI, DNA-bağlayıcı. DAPI Ölü hücrelerin plazma zarına (fakat canlı hücre) ve bağlama kromozom DNA geçebilir. </li><li> Süpernatantı aspire ve 100 ul HBSS-tam hücre pelletini. </li><li> anti-fare CD19-PE 1:40 seyrelti, anti-fare CD11 b-APC-Cy7 1:80 seyrelti, anti-fare PUKÖ 1-FITC 1:40 seyreltme ya: her bir karşılık gelen FACS tüpü içinde, aşağıdaki antikorların birisi ekleme manufa tarafından tavsiye edildiği gibi anti-fare B220-V500 01:40 seyreltmecturer. Oldukları gibi 5 inci (DAPI) ve 6. (boyasız) FACS tüpler bırakın. sallayarak iyice karıştırın ve 15 dakika boyunca karanlıkta buz üzerinde tüm FACS tüpleri kuluçkaya yatmaktadır. </li><li> İnkübasyondan sonra, 5 dakika, 4 ° C, 800 x g hızında, her bir tüp ve santrifüj içine FACS tüpler 4 mi buz gibi soğuk HBSS dolu ekleyin. </li><li> Santrifüj işleminden sonra, süpernatan aspire ve 500 ul buz hücre pelletini 500 ul FACS yükleme çözeltisi içinde tekrar süspansiyon haline getirilir DAPI işaretli tüp, pellet haricinde soğuk HBSS dolu yeniden süspanse edin. sıralama kadar karanlıkta buz üzerinde 6 tüpleri tutarak. </li></ol></li></ol> 6. Hücre Sıralayıcısı üzerinde sıralama NOT: sitometresindeki ve yazılım prosedürü sıralama Operasyonu şirket tarafından sağlanan ayrıntılı bir talimat ile standardize edilmiştir. Kısaca, biz 20 psi 100 mikron nozul, 5 arasında ayarlanabilir hedef hücre konsantrasyonunu kullanmak - ml başına 10 milyon ve% 70 ya da daha yüksek verimlilik ayarlamak (yani, çatışmalar 30 yaşın altında tutulan%). <ol><li> 100 U / ml penisilin-streptomisin içeren 500 ul FBS / tüp ile toplanması FACS tüpü hazırlayın. </li><li> Adım 5.2 den tek leke kompanzasyon tüpleri kullanılarak hücre sıralayıcı tazminat parametrelerini ayarlayın. <ol><li> Her florasan yoğunluğu için negatif bir kapı ayarlama boyanmamış bir tüp içinde 10,000 hücre kaydedin. </li><li> Her floresan yoğunluğu için pozitif kapısı ayarlamak için diğer tek leke tazminat tüplerde 10.000 hücreleri kaydedin. NOT: Yazılım otomatik olarak tazminat parametrelerini hesaplar. </li></ol></li><li> 3000 hücrelerini kaydederek sıralı hücre popülasyonlarının kapılarını ayarlamak için adım 5.1 bir örnek kullanın. el görünüşe göre diğer noktalardan ayrı noktalar grubu olarak X ve Y ekseni grafik geçit hedef hücre popülasyonunun parametre olarak floresan yoğunluğunu kullanılması. NOT: Bu yolluk seti araştırmacıların ihtiyaçlarına göre esnek ve keyfidir. Araştırmacılar, bir ya da daha fazla belirteçler dayalı yüksek saflıkta bekliyorsanız, kapı hareketyüksek gelen floresan yoğunluğuna göre. </li><li> Bir koleksiyon tüp yükleyin ve hedef hücre popülasyonunu sıralamak başlar. </li><li> Tüm örnek tüpleri yapılır kadar sıraladıktan sonra buz üzerinde toplama tüpü tutun. </li><li> 4 ml, sınıra kadar toplama tüpüne buz C10 ekleyin ve karıştırın ters. </li><li> 5 dakika, 4 ° C için 800 xg&#39;de toplama tüpü Santrifüj ve sonra el değmemiş hücre pelet ile yaklaşık 200 ul bırakarak süpernatant aspire. </li><li> hücre pelletini tekrar süspansiyon ve 1,5 ml santrifüj tüpüne Tüm hücre süspansiyonu transfer 1 ml buz gibi soğuk C10 ekleyin. </li><li> Santrifüj bakir hücre peleti 100 ul bırakarak 1.5 mi, 5 dakika, 4 ° C, 800 x g hızında boru ve süpernatan aspire. </li><li> kullanılana kadar buz üzerinde sıralanmış hücre popülasyonlarının saklayın. </li></ol>

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The authors declare that there is no conflict of interest regarding the publication of this paper.

The protocol described in this manuscript is for high purity enrichment of live pDC that retain the ability to produce IFN&#945;. The applications of this protocol include, but are not limited to, purification of pDC and/or any other mononuclear cells from the bone marrow of MRL/lpr and any other mouse strains for studies of cellular and molecular functions. Several critical steps in this protocol are to ensure high viability and purity of the sorted pDC. The first key step is the release of bone marrow from bones. To mi...

Efficient separation of a cell population of choice from other cells enables studies of the population that may not be possible otherwise. Fluorescence-activated cell sorting (FACS) is a method to enrich an interesting cell population with high purity. 1,2 Different cell types usually express unique molecules, or a unique combination of several molecules, on the plasma membrane that can distinguish one cell population from another. Upon binding of these cell surface molecules by specific fluorescence-conjugated antibodies, a detecting machine called flow cytometer/sorter is able to excite and detect the light signals of different fluorescent dyes that represent different molecule markers on the cells at the single cell level. The combined information consisting of either the presence of a light signal (representing positive expression of the corresponding surface molecule) or the absence of a light signal (representing negative expression of a molecule) defines the phenotype of the cell. After passing through the detector, cells with the same phenotype of interest are diverted towards a designated collecting tube based on electrical charge.
FACS is broadly applied in various studies as long as the population to be enriched is labeled with fluorescence.3-7 It has been used to separate immunoglobulin (Ig)A-coated bacteria from non-IgA coated bacteria in the gut microbiota 8 and sort genetically engineered cell populations expressing fluorescent proteins. 9 Importantly, it has the capacity to separate more than one population simultaneously, which not only saves time and reagents but also allows for more sophisticated study designs. 10 However, FACS also has its limitations. If a population of interest is very rare (less than 1%), the sorting efficiency may be reduced, causing significant cell loss. In addition, some antibody binding may activate intracellular signal transduction that induces functional changes of the sorted cell population. 11 Therefore, the phenotype used for sorting should be selected carefully. 
Other methods exist besides FACS that are also based on cell surface markers for the enrichment of specific cell populations, such as magnetic-activated cell sorting (MCS). 12 Similar to FACS, magnetic beads-conjugated antibodies can target specific cell surface molecules. Upon antibody-antigen interaction, magnetic beads-coated cells can be separated from non-coated cells after passing through a magnetic field. However, only a limited number of molecules can be targeted in MCS, as magnetic beads are, unlike various fluorescent colors in FACS, undistinguishable. It is thus difficult for MCS to define a cell phenotype with a complicated combination of surface markers. 13,14 In addition, MCS is also able to cause unintended activation of target cells.
In our studies of a mouse model of systemic lupus erythematosus (SLE), 15 we intended to purify plasmacytoid dendritic cells (pDC) to investigate their functional changes with disease progression. We first used MCS to enrich pDC from the bone marrow by targeting PDCA-1, a molecule highly and uniquely expressed on murine pDC at steady state. 16 However, the cell purity was unexpectedly low, likely due to the upregulation of PDCA-1 on other cell populations in an inflammatory environment such as SLE.16 Ultimately, we have used FACS with a combination of four surface markers (CD11c, CD11b, B220 and PDCA-1) to separate high-purity pDC as CD11c+CD11b-B220+PDCA-1+ population. Murine pDC has another specific surface marker Siglec-H. We decided not to use Siglec-H, as antibody binding of this molecule represses the function of pDC to produce IFN&#945;. 11

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>HyClone</td><td>SH30396.03</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>1x Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>gibco by life technologies</td><td>14175-079</td><td></td></tr><tr><td>MACS BSA Stock Solution</td><td>Miltenyi Biotec</td><td>130-091-376</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>SIGMA</td><td>M8266</td><td></td></tr><tr><td>DNase I</td><td>SIGMA</td><td>D4527</td><td></td></tr><tr><td>Red blood cell (RBC) lysis buffer&amp;nbsp;</td><td>eBioscience</td><td>00-4300-54</td><td></td></tr><tr><td>Density gradient medium</td><td>GE Healthcare</td><td>17-1440-02</td><td>Ficoll-Paque Plus&amp;nbsp;</td></tr><tr><td>anti-mouse CD19-PE</td><td>BD Pharmingen</td><td>553786</td><td></td></tr><tr><td>anti-mouse CD11c-PE</td><td>eBioscience</td><td>12-0114-82</td><td></td></tr><tr><td>anti-mouse CD11b-APC-CY7</td><td>BD Pharmingen</td><td>557657</td><td></td></tr><tr><td>anti-mouse PDCA-1-FITC</td><td>eBioscience</td><td>11-3172-81</td><td></td></tr><tr><td>anti-mouse B220-V500&amp;nbsp;</td><td>BD Pharmingen</td><td>561226</td><td></td></tr><tr><td>DAPI</td><td>invitrogen</td><td>D3571</td><td></td></tr><tr><td>Plasmacytoid Dendritic Cell Isolation Kit II, mouse</td><td>Miltenyi Biotec</td><td>130-092-786</td><td></td></tr><tr><td>BD FACSAria I flow cytometer&amp;nbsp;</td><td>BD Biosciences</td><td>643178</td><td></td></tr><tr><td>BD FACS Diva version 6</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

fluorescence-activated cell sorting for purification of plasmacytoid dendritic cells from the mouse bone marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

Biz yüksek saflıkta kemik iliği PDC zenginleştirmek amaçladık ve IFNa üretme kabiliyetleri konusunda pDC fonksiyonel değişiklikleri incelemek için genç ve yaşlılık hem MRL / lpr lupus eğilimli farelerin diğer hücre türleri etkisi olmadan. Kullanılan ilk arıtma stratejisi Şekil 1&#39;de görüldüğü gibi zenginleştirme sonra sadece% 7,75 saflıkta yol açtı, MCS, oldu. MCS ile karşılaştırıldığında, FACS% 96.4 gibi yüksek saflıkta PDC zen...

Watch this Scientific Journal Video about Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow at JoVE.com

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Biz pDC fonksiyonel çalışmalar için, lupus-eğilimli farelerin kemik iliğinden, yüksek saflıkta plazmasitoid dendritik hücreleri (PDC) izole etmek için sıralama floresans ile aktive edilmiş hücre kullanarak bir protokol sunulmuştur.

Fare kemik iliğinden plazmasitoid dendritik hücreleri Saflaştırılması floresans ile aktive edilen hücre sınıflandırma

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

fluorescence-activated-cell-sorting-for-purification-plasmacytoid

Research

JoVE Journal

Immunology and Infection

12.2K Görüntüleme.  Virginia Polytechnic Institute and State University.  Biz pDC fonksiyonel çalışmalar için, lupus-eğilimli farelerin kemik iliğinden, yüksek saflıkta plazmasitoid dendritik hücreleri (PDC) izole etmek için sıralama floresans ile aktive edilmiş hücre kullanarak bir protokol sunulmuştur. 

Video: Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Floresan Aktive Hücre Ayırma (FACS) Özgül Hücre Nüfus Arıtma

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

İmmünoloji ve Enfeksiyon

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Fare Akciğer Dendritik Hücrelerinin İzolasyonu

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

Akım Sitometri ve Otomatik Manyetik Aktif Hücre Sıralama (AutoMACS) kullanarak Naive ve Pankreas Tümör taşıyan fareler ile myeloid türevi bastırıcı hücreler (MDSC) hazırlanması

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

Tıp

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Fare Barsak ayrı dendritik hücreler ve makrofajlar İzolasyonu ve Karakterizasyonu

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

İnsan Thymus ile myeloid dendritik hücreleri ve epitel hücreleri İzolasyonu

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Hematopoetik progenitörlerin Evlatlık Transferi kullanarak Peter Parçacıklannda Murin plazmasitoid dendritik hücreleri Gelişimini değerlendirilmesi

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

AC-6 Besleyici Sistemi kullanılarak Ortak Lenfoid progenitörlerin dan Fare plazmositoid dendritik hücreler İn Vitro Nesil

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Fare Dendritik Hücre Alt Grupları İzolasyonunda bir Verimli ve Yüksek Verim Yöntem

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Kemik İliği Kaynaklı Dendritik Hücrelerin Farelerden İzole Edilmesi ve Kültürlenmesi için Ekonomik ve Verimli Protokol

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Source: Chang, S., et al. <a href="https://app.jove.com/v/53211">In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with &#947;-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

Ortak Lenfoid Progenitörlerden Plazmasitoid Dendritik Hücrelerin İn Vitro Üretimi

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. ...