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

<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.

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

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.

マウス肺樹状細胞の分離

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.

フローサイトメトリーおよび自動化された磁気活性化細胞選別（AutoMACS）を使用して、ナイーブ膵腫瘍を有するマウスからミエロイド系のサプレッサー細胞（MDSC）の調製

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

医学

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.

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

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.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

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.

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 ...

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 ...

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.

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

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.

マウスから骨髄由来樹状細胞を単離および培養するための経済的かつ効率的なプロトコル

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.remotexs.ntu.edu.sg/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.

一般的なリンパ系前駆細胞からの形質細胞様樹状細胞のin vitro作製

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