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.

注：MRL / MP-のFas のlpr狼瘡傾向マウスを繁殖させたとバージニア工科大学（動物福祉保証番号での施設内動物管理使用委員会（IACUC）の要件以下の特定病原体フリーの施設で維持した（MRL / LPR）： A3208-01）。この研究は、米国立衛生研究所の実験動物の管理と使用に関するガイドの推奨事項に厳密に従って行きました。全ての動物実験は、IACUCプロトコル番号12から062で行いました。 1.細胞培養培地およびソート・バッファ<ol><li>完全な細胞培養培地（C10）を10％ウシ胎児血清、1mMピルビン酸ナトリウム、1％の100 MEM非必須アミノ酸、10mMのHEPES、55μM2-メルカプトエタノール、2mMのL-グルタミンを補足したRPMI1640を使用して調製100 U / mlのペニシリン - ストレプトマイシン。 </li><li>並べ替えバッファを準備し、10mMのHEPESを補充した1×ハンクス平衡塩類溶液（HBSS）を用いて（フルHBSS）、2.5 mg / mlウシ血清アルブミン、0.05のMgCl 2および0.2 U / mlのDNアーゼI. </li></ol> 2.マウスの解剖<ol><li> 4ミリリットルのC10を含む2つの6 cmの直径の料理を準備します。氷の上に皿を置きます。 </li><li> CO 2吸入によりマウスを安楽死させます。 </li><li>脾臓を採取し、氷上でC10と皿の中でそれを保ちます。 <ol><li>解剖板に上向きに腹でマウスを突き止めます。 70％エタノールを噴霧することにより本体を滅菌。 </li><li>皮膚をカットし、下に首に恥骨結合から腹側正中線に沿って筋肉の壁から皮膚を分離します。 </li><li>足首への最初の切開部から後肢に沿って皮膚をカット。 </li><li>裏面に肌をピンとダイヤフラムに最初の切開場所から側面に沿って筋肉壁をカット。 </li><li>後頭部の右側の筋肉壁をピン。 </li><li>小腸の下、胃の横にマウスの右側に脾臓を探します。目の一端をピックアップ電子脾臓と胃から分離。 </li></ol></li><li>大腿骨と脛骨を含む、両方の後肢を切り取り、そしてできるだけ多くのすべての筋肉を取り除きます。 <ol><li>血管を回避しようとすると、鋭いハサミで足首に骨盤/股関節から後肢に沿って筋肉をカットします。 </li><li>骨髄内容の暴露せずに骨盤/股関節と足首/足関節で切断することにより、後肢を分離。 </li><li>繰り返しかみそりの刃で傷や関節のポイントで筋肉を切断することにより、骨の残りの筋肉を清掃してください。 </li></ol></li><li>氷の上で4ミリリットルのC10を含む6センチ皿に骨を保管してください。次のマウスを解剖に進んでください。 </li></ol> 3.脾細胞の単離<ol><li>赤い片が見えなくなるまで4ミリリットルのC10を含む皿の上に70μmのセルストレイナーに対するシリンジプランジャで軽く脾臓をホモジナイズします。 </li><li>ストレーナAを介して皿に追加の6ミリリットルのC10を追加します。NDその後、15ミリリットルコニカルチューブに合計10ミリリットルの細胞懸濁液を移します。 </li><li> 5分間遠心800×gで、4℃でコニカルチューブ。 </li><li>遠心分離後、上清を吸引し、2mlの1×赤血球（RBC）溶解緩衝液中で細胞ペレットを再懸濁します。室温で5分間インキュベートします。 </li><li>インキュベーション後、5分間、800×gで4°Cをコニカルチューブを遠心します。 </li><li>遠心分離後、上清を吸引し、1mlのC10に細胞ペレットを再懸濁します。任意の大きな塊（死細胞）を破棄し、後でチューブを氷上に保ちます。 注：多くの小さな塊がある場合は、一度細胞懸濁液をフィルタリングするために70μmのセルストレイナーを使用しています。 </li><li>自動細胞カウンターを用いてトリパンブルーを用いて細胞数をカウントします。 </li></ol> 4.骨髄細胞の単離<ol><li>乳鉢に4ミリリットルのC10と骨を移し、合計で10ミリリットルC10の容量を作るために6ミリリットルC10を追加します。 </li><li> APを使用して乳鉢で軽く骨をクラックestle。 </li><li> C10に骨髄を解放するために乳棒で軽くかき混ぜます。 </li><li> 70μmのセルストレイナーを通して氷上で50ミリリットルコニカルチューブに骨髄を含む10ミリリットルのC10を転送します。まだ骨を含む乳鉢に10ミリリットルの新鮮なC10を追加します。 注：ピペット赤い塊が観察される場合は、ストレーナを通過する前に数回上下に骨髄を含む溶液。 </li><li>繰り返し二回4.4から4.2を繰り返します。最後の洗浄後、骨は白表示されます。 </li><li> 5分間遠心800×gで、4℃で50ミリリットルコニカルチューブ。一方、RTで15 mlのコニカル遠心チューブ中の5 mlのにすぐに使用密度勾配培地を調製。 </li><li>遠心分離後、上清を吸引し、10mLのC10で細胞ペレットを再懸濁します。 </li><li>ゆっくり二相の間に明確な界面を維持5mlの密度勾配媒体の上部と10 mlのCell懸濁液を層。その後で30分間1363グラムで15ミリリットルコニカルチューブを遠心分離9と0（またはブレーキOFF）に設定し、減速としての加速度が設定されたRT（20℃）。 </li><li>遠心分離した後、慎重にトップ8ミリリットルの溶液を除去し、新しい15ミリリットルコニカルチューブにインタフェース（単核細胞の層）で2ミリリットルの軟膜を収集します。 </li><li>骨髄単核細胞、キャップを含む15ミリリットルコニカルチューブに12ミリリットルの氷冷C10を追加して、よく混ぜ、10分、4℃、800×gでチューブを遠心分離する数回転倒。 </li></ol> FACS 5.細胞表面染色<ol><li> サンプル染色 <ol><li>抗マウスCD16 / 32抗体溶液（HBSS-フルで1〜100希釈、5μg/ mlの最終濃度）を準備します。 </li><li>ステップ4.10において遠心分離後、上清を吸引し、単核細胞上のFc受容体をブロックするために100μlの抗マウスCD16 / 32抗体溶液中で細胞ペレットを再懸濁します。氷上で10分間インキュベートします。 </li><li>蛍光標識抗体の混合物を準備する（アリI-マウスのCD11c-PE 1：40希釈、抗マウスのCD11b-APC-CY7 1:80希釈、抗マウスPDCA-1-FITC 1：40希釈および抗マウスB220-V500 HBSS-で1：40希釈サンプルあたり100μlの最終容量で、製造業者によって推奨されるようにフル）。 注：これは、使用する前に抗体を滴定することが重要です。 </li><li>ステップ5.1.2での10分間のインキュベーション後、抗マウスCD16 / 32、非結合除去するために5分、4℃で800×gで4 mlの氷冷HBSSフルと遠心を追加します。 </li><li>遠心分離後、上清を吸引し、100μlの蛍光標識抗体の混合物で細胞ペレットを再懸濁します。暗所で15分間氷上でインキュベートします。 </li><li> 15分間のインキュベーション後、未結合の蛍光標識抗体を除去するために、5分間800×gで、4℃で4 mlの氷冷HBSSフルと遠心を追加します。 </li><li> FACSのローディング溶液（HBSS-フルで500 ngの/ mlのDAPI）を準備します。 </li><li>ステップ5.1.6で遠心分離した後、上清を吸引し、500μの細胞ペレットを再懸濁;リットルFACSローディング溶液。 12×75ミリメートルの丸底チューブ（FACSチューブ）に細胞懸濁液を移し、1時間以内に仕分けするまで暗所でチューブを氷上に保ちます。 </li></ol></li><li> 補償のための染色 <ol><li> PE、APC-CY7、FITC、V500、DAPIまたは未染色のようなラベル6は、FACSチューブ。一定分量のFACSチューブに1×10 6細胞/チューブで脾細胞を、5分間、4℃で800×gで4ミリリットルHBSS-フルで洗浄します。 注：使用してDNA結合蛍光色素、DAPI、死細胞から生細胞を区別します。 DAPIは（ただし生細胞）の死細胞の原形質膜を通過し、染色体DNAに結合することができます。 </li><li>上清を吸引し、100μlのHBSS-完全に細胞ペレットを再懸濁します。 </li><li>抗マウスCD19-PE 1：40希釈、抗マウスのCD11b-APC-CY7 1:80希釈、抗マウスPDCA-1-FITC 1：40希釈または：対応する各FACSチューブでは、以下の抗体の1を追加します。 manufaによって推奨されるように抗マウスB220-V500 1：40希釈cturer。そのまま5 回目 （DAPI）と6 番目 （未染色）FACSチューブを残します。振とうしてよく混合し、15分間、暗所で氷の上のすべてのFACSチューブをインキュベートします。 </li><li>インキュベーション後、各試験管に4 mlの氷冷HBSS-フルを追加し、5分間、800×gで4°CをFACSチューブを遠心してください。 </li><li>遠心分離後、上清を吸引し、500μlのFACSローディング溶液中に再懸濁されたDAPI標識されたチューブ内のペレットを除き、500μlの氷冷HBSSフルで細胞ペレットを再懸濁します。ソートするまで暗所で氷の上のすべての6のチューブを維持します。 </li></ol></li></ol> 6.セルソーターでソート注：サイトメーターおよびソフトウェア上の手順をソートの動作は、同社が提供する詳細な命令で標準化されています。 （ すなわち 、競合が30下に保持ミリリットル当たり10百万円、70％以上に効率を調整する-簡単に言えば、我々は5の間、標的細胞濃度を設定し、20 psiで100ミクロンのノズルを使用します％）。 <ol><li> 100 U / mlのペニシリン - ストレプトマイシンを含む500μlのFBS /チューブを収集FACSチューブを準備します。 </li><li>ステップ5.2からの単染色補償管を使用して、セルソーターの補償パラメータを調整します。 <ol><li>各蛍光強度のために負のゲートを設定するために染色されていないチューブで10,000個の細胞を記録します。 </li><li>各蛍光強度のために正のゲートを設定するには、他の単一染色補償チューブで10,000細胞を記録します。 注：ソフトウェアが自動的に補正パラメータを算出します。 </li></ol></li><li> 3000細胞を記録することにより、ソートされた細胞集団のゲートを設定するためにステップ5.1から1のサンプルを使用してください。明らかに他のドットから分離したドット群として手動で、X及びY軸グラフのパラメータとして蛍光強度をゲート標的細胞集団使用。 注：このゲーティングセットは、研究者の要件に基づいて、柔軟かつ任意です。研究者は1つ以上のマーカーに基づいて、より高純度が予想される場合、ゲートを移動高い対応する蛍光強度に基づいて。 </li><li>コレクションチューブをロードし、標的細胞集団をソートするために開始します。 </li><li>すべての試料管が行われるまで、ソートした後、氷上でコレクションチューブを保管してください。 </li><li> 4ミリリットル、キャップまでのコレクションチューブに氷冷C10を追加し、混合するために反転。 </li><li> 5分、4℃で800×gでコレクションチューブを遠心し、その後そのまま細胞ペレットと約200μLを残し、上清を吸引除去します。 </li><li>細胞ペレットを再懸濁し、1.5ミリリットル遠心管にすべての細胞懸濁液を転送する1ミリリットルの氷冷C10を追加します。 </li><li> 5分間800×gで遠心分離し1.5mlチューブ、4°C、上清を吸引、手つかずの細胞ペレットに100μlのを残します。 </li><li>使用するまで氷上でソートされた細胞集団を保管してください。 </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 minimize damaging bone marrow cells, it is important to gently crack the bones and stir the pestle instead of smashing the bones. The second key step is the enrichment of mononuclear cells with density gradient medium. It is critical to establish a clear interface between the lower phase (density gradient medium) and the upper phase (cell suspension) while loading the cell suspension. The centrifugation condition is also critical for the formation of the buffy coat layer that contains mainly mononuclear cells. After centrifugation, bone debris, red blood cells and high-density neutrophils are excluded from the buffy coat layer and stay at the bottom of the tube. The third key step is incubating the cells with fluorescence-conjugated antibodies at a low temperature (on ice or 4 &#176;C). After the staining, it is also important to wash the cells extensively to ensure the specific antibody binding with minimal false positives or background signals easily detectable by the cell sorter. Finally, the single-stain compensation tubes are important and necessary for the adjustment of compensation parameters to ensure only the correct signals are measured and analyzed on the cell sorter. Besides using isolated splenocytes for the compensation, commercially available compensation beads can be an alternative if cells are limited. In this particular study, as we needed to enrich as many pDC as possible for further functional studies, to avoid bone marrow cell loss, we used splenocytes instead of bone marrow cells for the compensation. Also, it needs to be noted that surface marker like CD11c are expressed on rare populations, so they are not the best choice for compensation. Instead, an alternative marker expressed at a relatively high level on a major population can be used for compensation purpose as long as the antibody is conjugated with the same fluorescence dye. For example, in this study, CD19-PE is used to replace CD11c-PE in the compensation staining of splenocytes. Besides single color controls, fluorescence minus one (FMO) controls are important for beginners, especially to sort a population with markers not apparently separated from other populations. However, in this study, single color controls are adequate as pDC are a distinct population clearly defined and separated from other cells. In addition, we could set the gate slightly smaller than the actual population to improve purity.
In our studies, we only sorted a single population by FACS. In fact, this sorting method can be used broadly not only to enrich several different cell populations simultaneously, but also to isolate any interesting particle targets within a certain size range decided by the cell sorter, as long as they can be labeled with fluorescence. MCS is another cell enrichment method similar to FACS by principle that both are based on antibody detection of cell surface markers. Even though MCS is also widely used, it may not be efficient in various situations. For example, the phenotype of a cell population may change with the microenvironment in vivo or culture condition in vitro. MCS is prone to errors when the cell phenotype changes, as it usually detects only one surface molecule. On the contrary, FACS is based on the combination of several adjustable markers that reflect a more specific and accurate phenotype of a cell population in dynamic conditions. In addition, the gating strategy of FACS gives it a more sophisticated sorting ability, such as sorting Ly6C+ monocytes into Ly6Chigh and Ly6Cmedium subsets, which cannot be done with MCS. 
However, FACS has its limitations as well. As it separates cell populations according to the analysis of fluorescent signals, if the cells or particles of interest have high autofluorescence, it will be difficult to sort the targets. In addition, a designated professional staff is usually required to operate the cell sorter properly. Moreover, the binding of fluorescence-conjugated antibodies to their specific ligands on cells may change the function of sorted cells, affecting the outcomes of subsequent experiments. For example, Siglec-H as a pDC specific surface molecule can be used to sort pDC, but the binding of Siglec-H by its specific antibody has been demonstrated to suppress the ability of pDC to produce IFN&#945;. Therefore, selection of markers for cell sorting needs to be cautious and based on the purpose of the study. 
Furthermore, the high-throughput cell sorter is limited to sorting, with high efficiency, only target cells of certain percentage within the total cell population. In this study, we first used density gradient centrifugation to increase the percentage of pDC through removing red blood cells, high dense granulocytes and debris, which was a better choice than simply adding the red blood cell lysis buffer. If the population is so rare that is less than 1%, the sorting efficiency will drop with significant cell loss. Hence, extremely rare population can be enriched first by other methods, such as negative selection with MCS to remove some of the other cells, and then sorted by FACS to achieve high purity with minimal cell loss. In summary, FACS is an efficient and accurate method to enrich functional pDC from the mouse bone marrow with high purity.

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

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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.

我々は、IFNαを産生する能力についてのpDCの機能変化を研究するために老いも若き両方年齢のMRL / LPRの狼瘡傾向マウスから、高純度の骨髄のpDCを豊かにすることを目的とした、および他の細胞型の影響を受けません。使用された最初の精製戦略は、 図1に示すように、濃縮した後にのみ7.75パーセントの純度につながった、MCS、でした。 MCSと比較して、FACSは、96.4％の高純度でのpDC濃縮しました。高純度を確保するために、ステップバイステップのゲーティング戦略を行いました。 図2に示すように、単核細胞をゲーティングし、前方散乱（FSC）および側方散乱（SSC）のパラメータによって破片から分離しました。 FSC幅（FSC-W）FSC-高さ対 （FSC-H）とSSC-H対SSC-Wを使用して、単一の細胞が集合体によって産生される偽陽性シグナルを回避するために、ゲートしました。ライブ単一細胞は、その後、DAPIとしてゲートした-人口（DAPI +細胞が死んでいる、またはアポトーシス）。生きた単一細胞、のCD11c + CD11bのから-集団がゲートされた、からB220 + PDCA-1 +集団をさらにPDCとしてゲートされました。ソートされたpDCだけでなく、高純度のも機能的に正常でした。 図3に示すように、彼らは私たちの以前の出版物17からの許可を得て再印刷（のCpG刺激によりin vitroで IFNαを産生する能力を持っていました。 <img alt="図1" src="/files/ftp_upload/54641/54641fig1.jpg" /> MCSおよびFACS方法のいずれかを介してのpDCソートの純度 1.図のpDC（CD11cの+のCD11b - B220 + PDCA-1 +）の割合を示す図代表的なフローサイトメトリー。MCS（上）およびFACS（下）によってソートされた細胞では、それぞれ。 CD11c + CD11bの-人口は、総生細胞（左）からゲートされ、その後、B220 + PDCA-1などのpDCました<html>人口（右） - 。+細胞は、CD11cの+のCD11b内でゲートされた<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54641/54641fig1large.jpg" target="_blank">本図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54641/54641fig2.jpg" /> FACSによるpDCを濃縮するための 図2 のゲーティング戦略次の順序でステップバイステップのゲート戦略：単核細胞の単一細胞FSCに、単一細胞SSCに、DAPIへ-のCD11c + CD11bのに生きている細胞、 - 、とします。 B220 + PDCA-1 +ライブPDCとして。総単一細胞内のPDCは2.95±1.42パーセントです。ソートされたpDCの数は、回収率約50％のマウス当たり0.88±0.28×10 5です。 （N = 12 MRL / lprマウス、データは平均値の平均±SD 95％CIとして示されています）。<html> 641fig2large.jpg &quot;ターゲット=&quot; _空白 &quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図3" src="/files/ftp_upload/54641/54641fig3.jpg" /> 。、in vitroで 6時間、FACSソートのpDCソート骨髄のpDC によって 図3.IFNα 生産は、クラスAのCpG（5μMODN1585）で処理しました。同図において、「Y」は若い、6週齢のマウスを表します。 「○」は、古い、16週齢のマウスの略です。 MRLは、健康な対照マウスとMRLマウスの略です。 LPRは、狼瘡傾向マウスとしてMRL / LPRマウスの略です。培養上清中のIFNαの濃度をELISAで測定しました。 * P &lt;0.05、** P &lt;0.01、*** P &lt;0.001、一元配置ANOVA。データは、平均±平均の標準誤差（群あたりn = 3匹のマウス）として示されています。この図は、許可を得て免疫学のジャーナルで私たちの出版物から再生されます。17PLOAD / 54641 / 54641fig3large.jpg &quot;ターゲット=&quot; _空白 &quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。

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

私たちは、PDCの機能研究のための狼瘡傾向マウスの骨髄から高純度の形質細胞様樹状細胞（pDC）を単離するために蛍光活性化セルソーティングを使用してプロトコルを報告しています。

マウスの骨髄から形質細胞様樹状細胞の精製のために、蛍光活性化細胞選別

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.1K ビュー.  Virginia Polytechnic Institute and State University. 私たちは、PDCの機能研究のための狼瘡傾向マウスの骨髄から高純度の形質細胞様樹状細胞（pDC）を単離するために蛍光活性化セルソーティングを使用してプロトコルを報告しています。 

ビデオ: 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.

蛍光活性化細胞選別（FACS）によって特定の細胞集団の精製

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

免疫・感染学

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

追跡調査のためのQdot ®二ナノとマウス骨髄由来樹状細胞の世代とラベリング

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

骨髄移植によるマウス大腸炎における免疫および非免疫細胞からの遺伝子発現の機能的役割を差別化

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

機能研究のためのマウス骨髄好中球と養子移入実験の分離、精製とラベリング

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

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.

造血前駆細胞の養子移入を使用して、パイエル板におけるマウス形質細胞様樹状細胞の開発を評価

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

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

マウス骨髄単球の追跡<em&gt;インビボ</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

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フィーダシステムを使用してリンパ球系共通前駆細胞からマウス形質細胞様樹状細胞のインビトロ生成に

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

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

大量の骨髄前駆細胞と新規セルソーティング戦略を使用してマウスの骨髄から樹状細胞前駆体の生成

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

メジャー ナイーブ cd4 陽性 T 細胞の活性化、増殖と骨髄由来樹状細胞による th1 細胞分化をIn Vivoマウス モデル

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...