この作品は、アレルギーと国立感染症研究所（NIAID）、国立衛生研究所（NIH）、米国の学内研究部門によってサポートされていました。 すべてのマウスは、アレルギーと国立感染症研究所（NIAID）での実験動物ケア認定の動物施設の認定のためのアメリカ連合に維持され、実験動物のケアと使用のためのガイドで説明する手順に従い、収容されたNIAIDの動物のケアと使用委員会によって承認されたプロトコルの後援の下に。

1。マウス骨髄細胞の単離<ol><li>機関の動物ケア委員会が承認したプロトコルを使用して、マウスを安楽死させると70％エタノールで動物の表面にスプレーします。 </li><li>半ば腹部の皮膚の切開を行い、下肢をカバーする皮膚を含むマウスの遠位部から皮膚を除去。 </li><li>はさみを使って下肢から筋肉を切断して大腿骨頭を壊す避けながら慎重に、股関節から寛骨臼を脱臼。 </li><li>メスやハサミを使用して大腿骨と脛骨から残りの筋肉を取り出し、骨端を壊さないために膝関節運動のケアで脛骨から大腿骨を分離する。氷冷RPMI 10％FBS、1％ペニシリン/ストレプトマイシンを補充した1640 1Xを含むペトリ皿に骨を置きます。 </li><li>組織培養フードの下には、次の手順に進んでください。 neutrophiを避けるために、厳格な無菌技術を維持するために余分な予防措置を取るLの活性化。 </li><li>骨の表面からエタノールを洗い流すための氷冷滅菌PBS（ペトリ皿内）で3回洗浄し、その後の70％エタノール（シャーレ内）で各ボーンをすすぐ。 </li><li>清潔な滅菌ペトリ皿の中では、骨の骨端を切断し、それらを脇に保つ。 </li><li> 25ゲージ針とRPMI、10％FBSおよび2mM EDTAを補充し、100μmのを取り付けた50mlスクリュートップファルコンチューブに骨軸の両端から骨髄細胞をフラッシュで満たされた12 ccの注射器を使用してフィルタ。効率的に全ての細胞を除去するために、25ゲージ針を用いて骨の内側表面をこすり。 </li></ol>注：骨のブランチングは、細胞が十分に削られていることを示しています。 注：大腿骨/脛骨ペアをフラッシュするために、メディアの約10ミリリットルを使用してください。媒体へのEDTAを添加すると、細胞の凝集を防ぐことが不可欠である。 <ol start="9"><li>骨骨端をカットメスで小0.5〜1ミリメートル3個および2.5ミリリットルエッペンドルフCombitipプラスBiopurピペットチップのバックエンドを使用して100μmのフィルターを介してそれらを破る。 </li><li> 4℃で7分間1,400 rpmで遠心</li><li>約20秒間のNaCl 0.2％20mlに細胞ペレットを再懸濁することにより、赤血球を溶解、1.6％のNaClを20ml加えた。 （クリティカル：骨髄細胞死を回避するために低張溶解の20〜30秒を超えないように、後者は好中球を活性化する可能性を秘めているので、溶解のために低張塩化ナトリウムの使用はACK溶解バッファーに推奨されます。） </li><li> 4で1,400 rpmで7分間遠心℃で細胞を収集する。 </li><li> 10％FBSおよび2mM EDTAを添加したRPMI 1640 1Xで細胞を洗浄し、ステップ1.12のように遠心分離。 </li><li>この方法を用いて骨髄細胞の収率は約60から80000000まで非感染8-12週齢のC57BL / 6マウス当たりである。 </li></ol> 2。好中球の分離密度勾配遠心分離によって<ol><li>骨髄細胞をカウントし、氷冷滅菌1mlのPBSに再懸濁する。 </li><li> 15 mlコニカルチューブにHISTOPAQUE 1119 3mlの（密度、1.119グラム/ ml）を追加します。 </li><li> HISTOPAQUE 1119 3mlの上HISTOPAQUE 1077のオーバーレイ3ミリリットル（密度、1.077グラム/ ml）を。 </li></ol>注：1119年と1077 HISTOPAQUE HISTOPAQUE℃を使用する前に18-26に温めなければならない。 重要なステップ：事前に勾配の準備として使用直前に勾配を準備するには、2つの層と、次善の好中球の純度と回収の間の拡散になります。 重要なステップ：遠心分離中の細胞分離を排除する2つの密度を混合することを避けるために徐々に行われるように1119年のニーズ以上のオーバーレイHISTOPAQUE 1077、。 <ol start="4"><li> HISTOPAQUE 1077の上に骨髄細胞懸濁液を重ねる。 </li></ol>重要なステップ：Overl細胞および1077 HISTOPAQUEの間の界面を乱すことを避けるためにゆっくりと行わなければならHISTOPAQUE 1077ニーズ上で骨髄細胞懸濁液をアイイング。 注：1mlのPBSで非感染マウスからの骨髄細胞を再懸濁することが&gt; 90％の純度好中球が得られる。しかし、多くの骨髄サンプルをプールすると好中球純度が損なわれ、例えば、3で300×10 6個の細胞を再懸濁mlのPBSは、&gt; 90％〜80％好中球純度を低減します。したがって、研究者は彼らの特定の実験のために理想的な細胞数/ボリュームの条件を識別するためのパイロット実験を行う必要があります<ol start="5"><li> 25℃で2,000 rpmで30分間、°ブレーキなしC遠心。 </li></ol>注：室温で勾配の遠心分離は、クリティカルおよび好中球の効果的な分離のために不可欠です。 <ol start="6"><li> 1119 HISTOPAQUEとHISTOPAQUE 1077層の界面に好中球を収集します。 </李&gt; <li> 4で7分間1,400 rpmで10％FBS、1％ペニシリン/ストレプトマイシンおよび遠心℃までで補充1640 1X RPMIで二回収集した好中球を洗う</li><li>好中球をカウントし、その実行可能性を決定する。 </li></ol>注：好中球は、典型的には&gt; FACS分析により測定した95％の生存及び&gt; 90％純粋。感染していない8-12週齢のC57BL / 6マウスの骨髄からの好中球の典型的な収量（ すなわち 2大腿骨と2脛骨の骨）〜6から12000000細胞です。好中球は、感染した動物の骨髄から採取されている場合、この番号は、実質的に大きい。 カンジダ感染マウスの細胞採取6のために使用されたときしたがって、〜30から40000000好中球/マウスを回収した。 3。 CellTracker染料を使用した好中球のラベル<ol><li> 37℃で温め℃のPBSで5×10 6細胞/ mlで好中球を再懸濁し</li><li>最後のコンセントでCellTracker染料を追加5μMの配給。 </li></ol>注：CellTrackerグリーン（CMFDA（5 - Chloromethylfluoresceinジアセテート）およびCellTrackerオレンジ（CMTMR（5 - （および-6） - 4 - クロロベンゾイルアミノテトラメチルローダミン）は、示差野生型および遺伝子欠損から好中球を標識するために、このプロトコルで使用されたマウス。 注：実験の日まで、-80℃で10 CellTrackerグリーンのMMとCellTrackerオレンジ、アリコート、店舗の原液°Cを準備します。 <ol start="3"><li>暗闇の中で振盪水浴中で37℃で10分間対応CellTracker色素の5μMと好中球をインキュベートする。 </li><li>氷冷RPMI 10％FBS、1％ペニシリン/ストレプトマイシンを添加した1640 1Xで細胞を2回洗浄します。 </li></ol>注：細胞の効率的な洗浄CellTracker色素で標識のステップはダウンのために差動で標識した好中球集団を混合する前に染料交差汚染を回避することが不可欠である後TREAM競争再増殖研究。 4。フローサイトメトリーを使用して転送好中球のマウスおよび分析における好中球の養子移入<ol><li> 25×10 6細胞/ mlと5×10 6好中球がマウスごとに転送されるように、横方向の尾静脈に懸濁液を200μlを注入の濃度で氷冷PBSで再懸濁し好中球。競争力の再増殖の好中球の研究のために、1:1の比率で、野生型および遺伝子欠損好中球を混ぜて、上記のようにマウス当たり5×10 6の好中球の合計を注入。 </li></ol>注：少なくとも、最大10×10 6好中球が養子動物に明らかな即時の毒性なしマウスごとに転送することもできる。 <ol start="2"><li>養子移入（ 例えば、1、2、3又は4時間後の転写）後異なる時間で、マウスおよび収穫血を安楽死させる、および/ ​​またはその他の骨髄および/ ​​または他の標的器官（単数または複数）。 </li><li> SIを準備公開プロトコル6,15を使用して養子移入ラベル好中球の定量的および定性的な分析のために、これらの組織からngle細胞懸濁液。 </li><li> LIVE / DEAD生存染色およびFcの封鎖、CD45（クローン30-F11）とラベル細胞Ly6G（クローン1A8）とのCD11b（M1/70クローン）とゲートライブCD45 + Ly6G +のCD11b +好中球上の次のこのゲートで好中球はFITC +（CellTrackerグリーンで標識されている場合）またはPE +（セルトラッカーオレンジで標識されている場合）であるレシピエントマウスのネイティブ好中球と同様、養子移入ラベル好中球を含む。 </li></ol>注：好中球は、少なくとも4時間後に転送するためにカンジダ感染マウスの血液、骨髄、腎臓で追跡することができる。 注：PBS中2％パラホルムアルデヒドで固定が悪影響CellTracker Grので標識された好中球の平均蛍光強度には影響を与えません少なくとも48時間EENまたはCellTrackerオレンジ。

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著者は、彼らが競合する利害関係を持たないことを宣言します。

本明細書で我々は、密度勾配遠心分離法を用いて高純度及び生存したマウスの骨髄からの好中球、多数の単離のための信頼できる、簡単、迅速かつ経済的なプロトコルを提示する。このプロトコルが正しく実行された場合、〜6-12×10 6の好中球は、感染マウスから回収することができ、などの多く〜として30〜40×10 6好中球は、感染後6マウスから単離することができる?...

好中球は、ヒトにおける最も豊富な白血球です。彼らは、微生物の侵入に対する防御の最初の行としての自然免疫系との行為の主な細胞成分である。好中球の数値および/ ​​または機能に影響を与える買収好中球減少症とプライマリ免疫不全患者では、宿主防御1でこれらの細胞の重要性を強調し、生命を脅かす侵略的な細菌や真菌感染症を開発。炎症組織2への血流から募集の好中球の走化が可能な勾配を発生させる化学誘引物質の分泌につながるオーケスト自然免疫応答の誘導においてそれらの同族パターン認識受容体の結果感染部位における侵入病原体の免疫認識。好中球は、感染部位を入力した後、彼らは、サイトカインやケモカイン産生につながる、病原体の取り込み活性化、及び酸化および非酸化を介して、私を殺してchanisms 3。先天性免疫におけるそれらのよく知られた役割に加えて、好中球はまた、最近効果的な適応免疫応答の開始剤4として重要な役割を果たすことが示されている。 5-7感染性および自己免疫疾患の様に示すように、一方、離間免疫において、保護の役割から、好中球はまた、過剰な蓄積および/ ​​または炎症部位 ​​において活性化に起因する組織損傷および免疫病理を媒介することができる。 いくつかの感染症や炎症状態に効果的な自然免疫応答とその多面的なエフェクター機能を搭載で好中球の不可欠な役割にもかかわらず、これらの細胞は、信頼性の高い実験プロトコルの欠如を取り扱うとの技術的な問題は、過去数十年にわたって好中球との研究を妨げている。したがって、好中球の単離のための再現可能なアッセイの使用は、好中球媒介immunoloに関する更なる研究を促進しなければならないgical機能エクスビボおよびin vivoである 。現在までに、いくつかの方法は、ヒト血液、マウス血液または骨髄8,9の密度勾配遠心分離、正または負の免疫マウスの血液または骨髄10,11から好中球を濃縮し、収穫などの好中球の単離のために記載されているチオグリコール酸または他の炎症剤12の腹腔内注射後のマウスの腹膜腔から好中球。好中球は簡単に人間の血液から大量に単離することができるが、この方法では、機能的な研究や養子移入実験13に十分な好中球の分離を排除マウス血液の限られた量のためにマウスでは次善です。腹膜腔から細胞を誘発チオグリコレートの収率は、マウスの血液に比べて大きくなるが、さらに、炎症性腹腔洗浄液中の好中球の純度の間で変化60から90パーセント、及び単離された好中球が活性化された表現型を示す。マウス腹腔12が定常状態でいくつかの好中球を有するこのように、この方法を用いて回収した細胞のみが、非刺激好中球の活性化ではなく、機能的研究を行うために使用することができる。その代わりに、骨髄は、そのような貪食、殺害や脱顆粒などの下流の機能研究のために、またはレシピエントマウスに養子移入に使用することができますどちらか刺激されていないか、または活性化された好中球11,14、大量の収穫のための便利な貯水池です。 ここで我々は、高収率を提供していますシンプルで高速な（〜2時間）プロトコルを記述（〜6-12×10 6好中球/非感染マウス、または最大30〜40×10 6好中球/感染したマウス）純粋な（80の-95％）骨髄から&gt; 95％の生存率で好中球。この方法は、密度勾配セルSEPAある市販のHISTOPAQUEを使用配給メディアはマウスの骨髄からの好中球を分離するために、フィコールナトリウムジアトリゾエートから成る。この方法は血液または腹膜腔に比べてマウス当たりの好中球のかなり大きな数字をもたらす、それは定常状態でのまたは感染後の両方のマウスから好中球を収集するために使用することができ、それは不連続使用して密度勾配遠心法と比較して、層に容易である55％/ 65％/ PBS 9で75％パーコール成るパーコール勾配。また、純粋な好中球を収集するのに必要な時間とリソースを大幅に蛍光活性化細胞ソーティングを用いた好中球の分離に比べて減少する。また、この方法は免疫濃縮工程を必要としないので、それはより費用対効果であり、したがって、好中球活性化の可能性を減少させる、磁気カラムおよび抗体への細胞の曝露を回避する。 単離されたneutrophの機能的研究を行うことに加えてILS のex vivoとレシピエントマウスへの細胞の養子移入は、このプロトコルは異なるCellTracker染料を使用して隔離された好中球を標識するための方法についても説明します。種々の遺伝的背景のマウスからの好中球の特異的標識は、血液から好中球の輸送における特定の遺伝子の直接的な役割に機械的な洞察を提供することができ、フローサイトメトリーを使用して、レシピエントマウスの組織において転送された好中球を追跡するために競争力の再増殖試験に適合させることができるターゲットに炎症臓器6。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>RPMI 1640 1X with L-glutamine and 25 mM HEPES</td>
 <td>Cellgro</td>
 <td>10-041-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (Heat inactivated)</td>
 <td>GemCell</td>
 <td>100500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)</td>
 <td>GIBCO</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M EDTA</td>
 <td>Quality Biological Inc</td>
 <td>351-027-101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.2% and 1.6% sodium chloride</td>
 <td>JT Baker</td>
 <td>3624</td>
 <td>Sodium chloride solutions prepared using distilled water and sterile filtered</td>
 </tr>
 <tr>
 <td>Sterile filtered Histopaque 1077</td>
 <td>Sigma</td>
 <td>10771</td>
 <td>Histopaque 1077 needs to be brought to 18-26 &deg;C before use</td>
 </tr>
 <tr>
 <td>Sterile filtered Histopaque 1119</td>
 <td>Sigma</td>
 <td>11191</td>
 <td>Histopaque 1119 needs to be brought to 18-26 &deg;C before use</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline (PBS) without Calcium and Magnesium</td>
 <td>Cellgro</td>
 <td>210-40-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethyl Alcohol (200 proof)</td>
 <td>The Warner Graham Company</td>
 <td>64-17-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CellTracker Green (CMFDA, 5-Chloromethylfluorescein Diacetate)</td>
 <td>Invitrogen</td>
 <td>C-7025</td>
 <td>Make stock solutions of 10 mM in DMSO, aliquot and store at -80 &deg;C</td>
 </tr>
 <tr>
 <td>CellTracker Orange (CMTMR, 5-(and 6)-4-Chloromethyl Benzoyl Amino Tetramethylrhodamine)</td>
 <td>Invitrogen</td>
 <td>C-2927</td>
 <td>Make stock solutions of 10 mM in DMSO, aliquot and store at -80 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-mouse CD45 (Clone 30-F11)</td>
 <td>eBioscience</td>
 <td>170451-82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse Ly6G (Clone 1A8)</td>
 <td>BD Pharmingen</td>
 <td>551461</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse Ly6G (Clone 1A8)</td>
 <td>BD Pharmingen</td>
 <td>560599</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse CD11b (Clone M1/70)</td>
 <td>eBioscience</td>
 <td>47-0112-82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse CD16/CD32 (Mouse BD Fc Block)</td>
 <td>BD Pharmingen</td>
 <td>553141</td>
 <td>Use at 1:100 dilution</td>
 </tr>
 <tr>
 <td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td>
 <td>Molecular Probes</td>
 <td>L-23105</td>
 <td>Use at 1:1000 dilution</td>
 </tr>
 <tr>
 <td>Dimethyl Sulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>67-68-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paraformaldehyde Solution, 4% in PBS</td>
 <td>USB Corporation</td>
 <td>19943</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table 1. Reagents for isolation, labeling and tracking of adoptively transferred neutrophils.</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>C57BL/6 mice</td>
 <td>Taconic</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes</td>
 <td>Corning</td>
 <td>430053</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml centrifuge tubes</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>25 ml serological pipettes</td>
 <td>Celltreat</td>
 <td>229225B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 ml serological pipettes</td>
 <td>Celltreat</td>
 <td>229210B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5 ml serological pipettes</td>
 <td>Celltreat</td>
 <td>229205B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pasteur pipettes (3 ml)</td>
 <td>BD Falcon</td>
 <td>357575</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>12 ml syringes</td>
 <td>Kendall monoject</td>
 <td>512878</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>25 G x 5/8 in. Needles (precision glide needles)</td>
 <td>BD</td>
 <td>305122</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 mm cell strainers</td>
 <td>BD Falcon</td>
 <td>352360</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bactericidal Petri dishes</td>
 <td>BD Falcon</td>
 <td>351029</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Combitips Plus Biopur</td>
 <td>Eppendorf</td>
 <td>2249608-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse dissecting instruments (Scissors, forceps, scalpel)</td>
 <td>Biomedical Research Instruments</td>
 <td>10-2300, 10-2165, 25-1200, 26-1000</td>
 <td>Instruments sterilized prior to use</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Tissue culture hood</td>
 <td>The Baker Company</td>
 <td>SG403</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Refrigerated centrifuge</td>
 <td>Thermo Fischer Scientific</td>
 <td>75004521</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>37 &deg;C shaking water bath</td>
 <td>Thermo Fischer Scientific</td>
 <td>3166721</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table 2. List of Materials and Equipment used in this protocol.</td>
 </tr>
 </tbody>
</table>

isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments

好中球は、自然免疫系の重要なエフェクター細胞である。彼らは急速に急性炎症のサイトで募集し、炎症環境に応じて、保護や病原性の効果を発揮している。それにもかかわらず、免疫における好中球の不可欠な役割にもかかわらず、別の感染症や炎症性の条件で好中球 &#39;エフェクターとimmunopathogenic効果を媒介分子の要因の詳細な理解は、まだ、一部ため、その短い半減期のために、これらの取り扱いとの難しさを欠いている細胞および下流機能的研究と養子移入実験のために好中球の十分な数を取得するための信頼性のある実験プロトコルの欠如。したがって、簡単、迅速、経済的かつ信頼性のある方法は、食作用、殺、サイトカイン産生、脱顆粒及び売買などの機能を評価するためにマウスの好中球の十分な数を収穫するために非常に望ましい。そのために、我々は、高純度と生存したマウスの骨髄からの好中球の多数を分離する任意の研究室で適応させることができる再現性の密度勾配遠心分離ベー​​スのプロトコルを提示。さらに、我々は、養子レシピエントマウスに移し、フローサイトメトリーを使用して少なくとも4時間後に転写するためのいくつかの組織で追跡することができる単離された好中球を標識するためにCellTracker染料を使用する簡単なプロトコルを提示する。このアプローチを使用して、異なるCellTracker染料、野生型および遺伝子欠損マウスからの好中球の特異的標識が正常にin vivoで標的組織への血液から好中球の輸送における特定の遺伝子の直接的な役割を評価するために競争力の再増殖試験を実行するために使用することができる。

このプロトコルは、マウスからの骨髄細胞と市販のHISTOPAQUE細胞分離媒体を用いた密度勾配遠心分離によりこれらの細胞から好中球のその後の分離を収穫するために最適化される。この方法を用いて単離された好中球は、下流の機能研究のex vivoでの様々なレシピエントマウスにおける養子移入実験のために使用することができる。 感染していない8-12週齢のC57BL / 6?...

Watch this Scientific Journal Video about Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments at JoVE.com

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

我々は、密度勾配遠心分離によりCellTrackerと染料を用いて好中球標識のマウス骨髄からの好中球の単離および精製のためのプロトコールを述べる。これは、下流の機能研究または養子移入と追跡実験の好中球の多数を取得するための、簡易で、迅速かつ再現性のある経済的な方法を表しています。

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 ...

isolation-purification-labeling-mouse-bone-marrow-neutrophils-for

Research

JoVE Journal

Immunology and Infection

61.3K ビュー.  National Institute of Allergy and Infectious Diseases, NIH. 我々は、密度勾配遠心分離によりCellTrackerと染料を用いて好中球標識のマウス骨髄からの好中球の単離および精製のためのプロトコールを述べる。これは、下流の機能研究または養子移入と追跡実験の好中球の多数を取得するための、簡易で、迅速かつ再現性のある経済的な方法を表しています。

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

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

実験的転移とCTLの養子移入免疫療法のマウスモデル

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

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 and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

単離とマウス骨髄由来の単球の静脈注射

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

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 ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

マウス後肢長骨解剖や骨髄の単離

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

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.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

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 ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

分離と機能研究の好中球由来の微粒子のキャラクタリゼーション

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

寡能性と系列拘束されたマウスの骨髄から骨髄前駆細胞の分離と同定

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.