作者は彼の技術支援のために張兵に感謝したいと思います。この作品は、健康の研究（MOP-86595）のためのカナダの協会によって資金を供給された。 TABはCIHR新治験責任医師とAHFMRの学者である。博士とAIHSフルタイム学生の身分 - QHはCIHRカナダ大学院奨学金によってサポートされています。 SANは、クイーン·エリザベスII大学院奨学金によってサポートされています。博士 - AYWSはNSERC大学院奨学金によってサポートされています。

実験プロトコルの全体的なスキームについては図1を参照してください。 1。解剖<ol><li> 60×15ミリメートルペトリ皿に置き、無菌スチールメッシュスクリーン。 1単位は、組織サンプルあたり必要とされている。 </li><li>各皿にハンクス平衡塩溶液（HBSS）5mlを加える。氷の上で料理をしてください。 </li><li> CO 2とマウスを安楽死させる。 </li><li>解剖表面へのセキュアなマウス、上向きに腹側。スプレーは殺菌のため70％エタノールでマウスや毛皮がダウンつや消しされていることを確認する。 </li><li>手術用のハサミを使用して、ちょうど性器上腹部皮膚に腹切開を行うことで、解剖を開始します。あごに切開を上向きに伸びる。 </li><li>正中線から、すべての手足に沿って切開を下に拡張します。たるんだ皮膚を引いて、切開面にそれを突き止める。 </li><li>胸腺の収穫： <ol><li>切開を作るために胸骨の下部先端を持ち上げます。</li><li>肝臓を避けて、胸郭をデタッチし、各側に胸郭をカットするダイアフラムを切った。肺と心臓を避けるように注意しながら、それぞれの側に胸郭を上方にカットします。 </li><li>胸郭バックピンセットで慎重に引き抜いてください。胸腺は心臓の上方に位置する白、両葉性臓器である。ローブの底を把握するために鉗子の平らなエッジを使用して、静かに胸腺を引き、メッシュスクリーンの上に置きます。 </li></ol></li><li>脾臓を回収します。 <ol><li>脾臓は肝臓下マウスの腹腔内の左側にある赤い、サーフボード形をした臓器です。 </li><li>腹腔内に切開を加えます。優しく結合組織を離れていじめるために第2のペアを使用して、鉗子の1ペアで脾臓を引き出します。別のメッシュスクリーンで脾臓を置きます。 </li></ol></li></ol> 2。細胞調製<ol><li> 3 mlのシリンジからプランジャーを使用して、挽く唯一の結合組織と脂肪が残るまでメッシュスクリーンに臓器。皿三回からHBSSでメッシュをすすぐ。各組織試料のための新しいプランジャーを使用しています。 <ol><li>組織を均質化するためのその他のオプションについては、このメソッドの代わりに使用することができます。 </li></ol></li><li>血球計数器を用いて細胞をカウントする。 </li><li> 4℃で5分間、335×gで℃で遠心分離により細胞をペレット化</li><li>室温で10分間、ACK溶解緩衝液（0.15M NH 4 Clを 、10mMのKHC0 3、0.1mMののNa 2 EDTA、pH7.2）を500μlの脾細胞を再懸濁します。 FACS緩衝液中で20×10 6細胞/ ml（PBS、1％FCS、0.02％アジ化ナトリウム）での胸腺細胞を再懸濁し、氷上に置いておきます。 </li><li> HBSS 5mlを加えて等張性に脾細胞を返します。 FACS緩衝液中で20×10 6細胞/ mlで再懸濁し、遠心分離によってペレットは脾。細胞は無菌を維持する必要がある場合は、滅菌したRPMI +10％FCSを使用することができます。 </li></ol> 3。フローサイトメトリー用の細胞の染色このプロトコルの目的は、野生型（WT）とHYのCD4マウスを用いた正と負の選択を分析するための戦略を概説することです。フローサイトメトリー実験のデザイン、収集、および分析に関する一般的な考慮事項については、我々は董らによる優れたレビューに読者を11 参照 <ol><li>アリコート4×10 96ウェルプレートの各ウェルに、フローサイトメトリー試料につき6胸腺細胞、ならびに補償制御当たり1×10 6 WT脾。トランスジェニックマウスを用いた場合は、実験のコントロールとして、WTマウスを含める必要があります。 </li><li>氷上で10分間anti-CD16/32（クローン2.4G2）で細胞をインキュベートすることにより、Fc受容体をブロックします。 </li><li> 5分間、4℃で335 xgで皿回しをする。蓋を外し、一度プレートをフリックして井戸から液体を払拭する、シンクに伏せ。 FACS緩衝液200μlの各ウェルを再懸濁します。洗浄を繰り返します。 </li><li>下記のようにウェル当たりのFACS緩衝液200μlに希釈に基づいて、各抗体の最適濃度を用いて、抗体カクテルを準備します。各抗体の最適濃度は、正と負の集団の最大の分離を与えるために必要な最低濃度として定義されています。所与の抗体には陰性集団が存在しない場合は、アイソタイプ抗体が含まれるべきである。必要に応じて、ウェルあたり総量（ウェルあたり100μlまでなど ）に縮小することができます。 <ol><li> WT胸腺：抗TCRβ、抗CD4、抗CD8、抗CD69または抗CD5、抗CD24 </li><li>胸腺HYのCD4：抗HY TCR（T3.70）、抗CD4、抗CD8、抗CD69または抗CD5、抗CD24 CD69 loと胸腺細胞におけるCD69 ハイテク集団のより良い分離を得るためには、続いて、ビオチン化抗CD69一次抗体を使用することが推奨される二次的な蛍光標識ストレプトアビジンを含む染色。私達はuCD5染色のためのSEが同じ戦略。 </li></ol></li><li>暗闇の中で氷上で30分間、FACSバッファーで抗体カクテル200μlで細胞をインキュベートします。 </li><li>抗体カクテル染色と同時に、単一蛍光標識抗体を用いて、各補償制御を染色。理想的には、補償染色は、抗体カクテルで使用したものと同じ抗体を含んでいる。多くの抗原がアッセイされたときしかし、個々の抗原を染色すると、大きな実験用可能でないかもしれません。したがって、抗CD4または抗CD8のどちらかに染色が高いこれらの抗原の発現、または補償ビーズの使用に起因することをお勧めします。暗闇の中で氷上で30分間FACS緩衝液で染色。 1補償制御は、汚れのないままにしておきます。 </li><li> FACS緩衝液で細胞を2回洗浄します。 </li><li> FACS緩衝液およびFACSチューブへの転送中に細胞を再懸濁します。 </li><li>フローサイトメーターでサンプルを取得。 FSC認証の取得とするFSC-Wを含むダブレット識別のためLLOW。 </li></ol> 4。フローサイトメトリーデータの解析 - 非TCRトランスジェニックマウス我々は、フローサイトメトリーデータ分析のためFlowJoを使用しています。非TCRトランスジェニックマウスのゲーティング戦略のために図2Aを参照してください。 <ol><li> SSCは、 &quot;リンパ球&quot;人口で電子的にゲートによって、FSC使用。 </li><li> &quot;リンパ球&quot;集団の中では、FSC-W loの人口は、細胞のダブレットとaggregratesを除外するために電子的にゲートにFSC-Wで、FSC使用。これは、 &quot;一&quot;の門です。 </li><li> &quot;一重&quot;ゲートは、CD4、CD8、プロット内のイベントを使用します。 DN、DP 鈍い 、DP 明るい 、CD4 + CD8 LO、CD4SPとCD8SP集団、図2Bに示されるようにするためのゲートを描画します。 </li><li> CD4SPとCD8SPゲートの下では、CD24のプロットによってTCRβを作成します。 図2Cに示すように、ゲートを描画する象限·ゲーティング·ツールを使用します。 </li><li> NEを作成&quot;一重&quot;ゲートからwプロット：CD69によってTCRβ。ゲートA、B、C、Dは、 図2Dに示すように描画します。 </li><li> CD5によってTCRβ： &quot;シングレット&quot;ゲートから別のプロットを作成します。 図2Eに示すように、ゲートI、II、III、IVを描きますこれは正の選択を調べるためのもう一つの戦略です。 </li><li>ゲートI、II、III、IV、A、B、C、D（ 図2D、E）に&quot;一&quot;の下からDN、DP 鈍い 、DP 明るい 、CD4 int型、CD4SPとCD8SPゲートを適用します。 </li><li>あなたのターゲット人口に到達するまで、後続の各ゲートの周波数によって臓器の細胞数を乗じて特定のサブセット内のセルの数を計算します。たとえば、TCRβ ハイテク CD69-CD8SP胸腺細胞の数は、 胸腺細胞数×％、TCR BHIによって決定されるであろうCD69-（画分D）×％のCD8SP。 <ol><li>生細胞と死細胞は、いわゆる &quot;lymphocが含まれていませんすでにカウントすることを考慮に入れあなたの計算でyte &quot;ゲート。 </li></ol></li></ol> 5。フローサイトメトリーデータの解析 - TCRトランスジェニックマウス TCRトランスジェニックマウスのゲーティング戦略のために図3Aを参照してください。 <ol><li>前節と手順4.1と4.2に従ってください。 </li><li> &quot;一重&quot;ゲートの下で、T3.70のSSCプロットとゲートでT3.70を作成+細胞集団を抗原特異的T細胞（ 図3B）を分析する。いくつかの状況では、それは非抗原特異的（T3.70-）T細胞集団に、この集団を比較するために興味があるかもしれません。 </li><li> T3.70 +集団内では、ドローのDN、DP CD4SPとCD8SPゲート（ 図3C）。 <ol><li> WTコントロールサンプルは、 &quot;一重&quot;ゲートの下、これらのゲートを描くか、非常に少数のT3.70 +細胞が存在するとしてT3.70ゲートを作成します。 </li></ol></li><li> CD8SPゲートの下に、作成CD24のプロットによってT3.70。 4つの集団（ 図3F）にセルを分割する象限·ゲーティング·ツールを使用します。 </li><li> WTマウスとHYのCD4マウスの T3.70 + DPコンパートメント（ 図4A）のDPコンパートメント内のCD69発現を描いオーバーレイヒストグラムを作成します。 CD5発現（ 図4B）を調べることを繰り返します。 </li><li> 4.8のように、目的の各サブセット内の細胞の絶対数を計算します。 </li></ol>

<ol>
	<li>Liston, A., Lesage, S., Wilson, J., Peltonen, L., Goodnow, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aire+regulates+negative+selection+of+organ-specific+T+cells.">Aire regulates negative selection of organ-specific T cells.</a> Nat. Immunol. 4, 350-354 (2003).</li><li>Liston, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+dosage--limiting+role+of+Aire+in+thymic+expression,+clonal+deletion,+and+organ-specific+autoimmunity.">Gene dosage--limiting role of Aire in thymic expression, clonal deletion, and organ-specific autoimmunity.</a> J. Exp. Med. 200, 1015-1026 (2004).</li><li>Hogquist, K. A., Baldwin, T. A., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+tolerance:+learning+self-control+in+the+thymus.">Central tolerance: learning self-control in the thymus.</a> Nat. Rev. Immunol. 5, 772-782 (2005).</li><li>Liston, A., Enders, A., Siggs, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unravelling+the+association+of+partial+T-cell+immunodeficiency+and+immune+dysregulation.">Unravelling the association of partial T-cell immunodeficiency and immune dysregulation.</a> Nat. Rev. Immunol. 8, 545-558 (2008).</li><li>Starr, T. K., Jameson, S. C., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+selection+of+T+cells.">Positive and negative selection of T cells.</a> Annu. Rev. Immunol. 21, 139-176 (2003).</li><li>McCaughtry, T. M., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+tolerance:+what+have+we+learned+from+mice.">Central tolerance: what have we learned from mice.</a> Seminars in immunopathology. 30, 399-409 (2008).</li><li>Zhan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Without+peripheral+interference,+thymic+deletion+is+mediated+in+a+cohort+of+double-positive+cells+without+classical+activation.">Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 1197-1202 (2003).</li><li>Brewer, J. A., Kanagawa, O., Sleckman, B. P., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocyte+apoptosis+induced+by+T+cell+activation+is+mediated+by+glucocorticoids+in+vivo.">Thymocyte apoptosis induced by T cell activation is mediated by glucocorticoids in vivo.</a> J. Immunol. 169, 1837-1843 (2002).</li><li>Martin, S., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-specific+and+nonspecific+deletion+of+immature+cortical+thymocytes+caused+by+antigen+injection.">Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection.</a> European journal of immunology. 27, 2726-2736 (1997).</li><li>Baldwin, T. A., Sandau, M. M., Jameson, S. C., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+timing+of+TCR+alpha+expression+critically+influences+T+cell+development+and+selection.">The timing of TCR alpha expression critically influences T cell development and selection.</a> J. Exp. Med. 202, 111-121 (2005).</li><li>Tung, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+flow+cytometry:+a+practical+approach.">Modern flow cytometry: a practical approach.</a> Clinics in laboratory medicine. 27, 453-468 (2007).</li><li>Aliahmad, P., Kaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+all+CD4+T+lineages+requires+nuclear+factor+TOX.">Development of all CD4 T lineages requires nuclear factor TOX.</a> J. Exp. Med. 205, 245-256 (2008).</li><li>Kastner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl11b+represses+a+mature+T-cell+gene+expression+program+in+immature+CD4(+)CD8(+)+thymocytes.">Bcl11b represses a mature T-cell gene expression program in immature CD4(+)CD8(+) thymocytes.</a> Eur. J. Immunol. 40, 2143-2154 (2010).</li><li>Albu, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BCL11B+is+required+for+positive+selection+and+survival+of+double-positive+thymocytes.">BCL11B is required for positive selection and survival of double-positive thymocytes.</a> J. Exp. Med. 204, 3003-3015 (2007).</li><li>Van De Wiele, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocytes+between+the+beta-selection+and+positive+selection+checkpoints+are+nonresponsive+to+IL-7+as+assessed+by+STAT-5+phosphorylation.">Thymocytes between the beta-selection and positive selection checkpoints are nonresponsive to IL-7 as assessed by STAT-5 phosphorylation.</a> J. Immunol. 172, 4235-4244 (2004).</li><li>Ueno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+signals+are+essential+for+cortex-medulla+migration+of+developing+thymocytes.">CCR7 signals are essential for cortex-medulla migration of developing thymocytes.</a> J. Exp. Med. 200, 493-505 (2004).</li><li>Saini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Zap70+expression+during+thymocyte+development+enables+temporal+separation+of+CD4+and+CD8+repertoire+selection+at+different+signaling+thresholds.">Regulation of Zap70 expression during thymocyte development enables temporal separation of CD4 and CD8 repertoire selection at different signaling thresholds.</a> Science signaling. 3, ra23 (2010).</li><li>Hu, Q., Sader, A., Parkman, J. C., Baldwin, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bim-mediated+apoptosis+is+not+necessary+for+thymic+negative+selection+to+ubiquitous+self-antigens.">Bim-mediated apoptosis is not necessary for thymic negative selection to ubiquitous self-antigens.</a> J. Immunol. 183, 7761-7767 (2009).</li><li>Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M., von Boehmer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+in+T-cell-receptor+transgenic+mice+involves+deletion+of+nonmature+CD4+8++thymocytes.">Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes.</a> Nature. 333, 742-746 (1988).</li><li>McCaughtry, T. M., Baldwin, T. A., Wilken, M. S., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+deletion+of+thymocytes+can+occur+in+the+cortex+with+no+involvement+of+the+medulla.">Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla.</a> J. Exp. Med. 205, 2575-2584 (2008).</li><li>Derbinski, J., Schulte, A., Kyewski, B., Klein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+gene+expression+in+medullary+thymic+epithelial+cells+mirrors+the+peripheral+self.">Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self.</a> Nat. Immunol. 2, 1032-1039 (2001).</li><li>Anderson, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+of+an+immunological+self+shadow+within+the+thymus+by+the+aire+protein.">Projection of an immunological self shadow within the thymus by the aire protein.</a> Science. 298, 1395-1401 (2002).</li><li>Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+class+I-restricted+exogenous+presentation+of+self+antigens+in+vivo.">Constitutive class I-restricted exogenous presentation of self antigens in vivo.</a> J. Exp. Med. 184, 923-930 (1996).</li><li>Nitta, T., Nitta, S., Lei, Y., Lipp, M., Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7-mediated+migration+of+developing+thymocytes+to+the+medulla+is+essential+for+negative+selection+to+tissue-restricted+antigens.">CCR7-mediated migration of developing thymocytes to the medulla is essential for negative selection to tissue-restricted antigens.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 17129-17133 (2009).</li><li>Bouneaud, C., Kourilsky, P., Bousso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+negative+selection+on+the+T+cell+repertoire+reactive+to+a+self-peptide:+a+large+fraction+of+T+cell+clones+escapes+clonal+deletion.">Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion.</a> Immunity. 13, 829-840 (2000).</li><li>Gallegos, A. M., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+tolerance+to+tissue-specific+antigens+mediated+by+direct+and+indirect+antigen+presentation.">Central tolerance to tissue-specific antigens mediated by direct and indirect antigen presentation.</a> J. Exp. Med. 200, 1039-1049 (2004).</li><li>Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+CD4(+)+T+cell+frequency+varies+for+different+epitopes+and+predicts+repertoire+diversity+and+response+magnitude.">Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude.</a> Immunity. 27, 203-213 (2007).</li><li>Bouillet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BH3-only+Bcl-2+family+member+Bim+is+required+for+apoptosis+of+autoreactive+thymocytes.">BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes.</a> Nature. 415, 922-926 (2002).</li><li>Suen, A. Y., Baldwin, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proapoptotic+protein+Bim+is+differentially+required+during+thymic+clonal+deletion+to+ubiquitous+versus+tissue-restricted+antigens.">Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens.</a> Proceedings of the National Academy of Sciences of the United States of America. , (2012).</li><li>Calnan, B. J., Szychowski, S., Chan, F. K., Cado, D., Winoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+orphan+steroid+receptor+Nur77+in+apoptosis+accompanying+antigen-induced+negative+selection.">A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection.</a> Immunity. 3, 273-282 (1995).</li><li>Zhou, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Nur77/Nurr1+leads+to+inefficient+clonal+deletion+of+self-reactive+T+cells.">Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells.</a> J. Exp. Med. 183, 1879-1892 (1996).</li><li>Baldwin, T. A., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+analysis+of+clonal+deletion+in+vivo.">Transcriptional analysis of clonal deletion in vivo.</a> J. Immunol. 179, 837-844 (2007).</li></ol>

特別な利害関係は宣言されません。

ここで紹介するプロトコルは、非トランスジェニックTCRおよびTCRトランスジェニックマウスにおける正と負の選択を調べるために使用することができます。このプロトコルは、表面抗原の染色を説明します。分子メカニズムの更なる分析のために、それはしばしば細胞内染色を行う必要がある。我々は、ほとんどの細胞内タンパク質と転写因子のBDバイオサイエンスのFoxp3の染色キット用BD Bio...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> Hyclone社ハンクス平衡塩溶液</td><td>サーモサイエンティフィック</td><td> SH30030.02 </td><td></td></tr><tr><td>メタルメッシュスクリーン</td><td> Cedarlane </td><td> CX-0080-J-01 </td><td></td></tr><tr><td>ペトリ皿（60×15ミリメートル） </td><td>フィッシャー·サイエンティフィック</td><td> 877221 </td><td></td></tr><tr><td>シリンジ（3ミリリットル） </td><td> BD Biosciences社</td><td> 309657 </td><td></td></tr><tr><td>コニカルチューブ（15ml）を</td><td>ザルスタット</td><td> 62.554.205 </td><td></td></tr><tr><td>顕微鏡</td><td>ツァイス - プリモスター</td><td> 415500-00XX-000 </td><td></td></tr><tr><td>血球計</td><td> Hausser科学的</td><td> 3110 </td><td></td></tr><tr><td> 96ウェルプレート</td><td>ザルスタット</td><td> 82.1582.001 </td><td></td></tr><tr><td>マルチチャンネルピペット</td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td>リン酸緩衝生理食塩水</td><td>フィッシャー·サイエンティフィック</td><td> SH3025802 </td><td></td></tr><tr><td>アジ化ナトリウム</td><td> ITベイカーケミカル株式会社</td><td> V015-05 </td><td></td></tr><tr><td> FcRブロッキング試薬</td><td></td><td></td><td>クローン2.4G2 </td></tr><tr><td>抗マウスHY TCR </td><td> eBioscience社</td><td> XX-9930-YY * </td><td>クローンT3.70 </td></tr><tr><td>抗マウスCD4 </td><td> eBioscience社</td><td> XX-0042-YY * </td><td>クローンRM4-5 </td></tr><tr><td> ANTI-マウスCD8α </td><td> eBioscience社</td><td> XX-0081-YY * </td><td>クローン53から6.7 </td></tr><tr><td>抗マウスCD24 </td><td> eBioscience社</td><td> XX-0242-YY * </td><td>クローンM1/69 </td></tr><tr><td>抗マウスTCRβ </td><td> eBioscience社</td><td> XX-5961-YY * </td><td>クローンH57-597 </td></tr><tr><td>抗マウスCD69ビオチン化</td><td> eBioscience社</td><td> 13から0691-YY * </td><td>クローンH1.2F3 </td></tr><tr><td>抗マウスCD5ビオチン化</td><td> eBioscience社</td><td> 13から0051-YY * </td><td>クローン53から7.3 </td></tr><tr><td>ストレプトアビジン</td><td> eBioscience社</td><td> XX-4217-YY * </td><td></td></tr><tr><td>フローサイトメーター</td><td> BD Biosciences社 - FACS Cantoの</td><td> 338962 </td><td></td></tr><tr><td> FACSチューブ</td><td> BD Biosciences社</td><td> 352052 </td><td></td></tr><tr><td>フローサイトメトリー解析ソフトウェア</td><td> TreeStar - Flowjo </td><td> FlowJo V7 / 9 </td><td></td></tr><tr><td> Hyclone社RPMI - 1640培地</td><td>サーモサイエンティフィック</td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> * XXは、蛍光色素とYYバイアルサイズによって異なりによって異なります。 </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

健康な免疫システムは、自己抗原に対する寛容を維持しながら、T細胞は外来抗原に反応している必要があります。 T細胞受容体（TCR）αおよびβ遺伝子座のランダム再配置は、自己と外国の両方に、抗原特異性の広大な多様性を有するT細胞レパートリーを生成します。胸腺での開発時にレパートリーの選択は、安全で有用なT細胞を生成するために重要です。胸腺選択の欠陥は、自己免疫疾患および免疫不全疾患1-4の発展に貢献しています。 T細胞の前駆細胞は、CD4またはCD8コレセプターを発現していないダブルネガティブ（DN）の胸腺細胞として胸腺を入力します。 αβTCRと両方共受容体の発現は、ダブルポジティブ（DP）の段階で発生します。胸腺細胞によって提示された自己ペプチド - MHCとαβTCR（pMHC複合）との相互作用は、DP胸腺細胞の運命を決定する。高親和性相互作用負の選択と消去又はにつながる自己反応性胸腺細胞のる。 CD4または自己MHCによって提示された5外来抗原を認識することができるCD8シングルポジティブ（SP）はT細胞の正の選択と開発における低親和性の相互作用の結果。 ポジティブセレクションは、成熟T細胞の生成を観察することによって、ポリクローナル（野生型）TCRレパートリーを持つマウスで検討することができます。しかし、彼らは小さな抗原特異的集団の削除を伴う負の選択の研究のために理想的ではありません。多くのモデル系は、負の選択を検討するが、生理学的事象6を再現する能力に変化させるために使用されてきた。外来抗原の投与が胸腺細胞7-9の非特異的欠失につながることができますたとえば、胸腺細胞のin vitroで刺激すると 、親密に選定に関与している胸腺環境を欠いている。現在、 生体ネガティブセレクションで勉強するための最良のツールはtransgを表現するマウスである自己抗原内因用enic TCRの特定。しかし、多くの古典的なTCRトランスジェニックモデルは時期尚早の負の選択で、その結果、DNの段階でトランスジェニックTCRα鎖の早期の発現によって特徴付けられる。私たちの研究室では、10野生型マウスで発生する負の選択がSP遷移にDPの中に発生することができ、トランスジェニックHYTCRαが条件付きでDPステージで表現されたHYのCD4モデルを開発しました。 ここでは、HYのCD4マウスモデルにおける胸腺の正と負の選択を検討するために、フローサイトメトリーベースのプロトコルを記述します。 HYのCD4マウスにおける負の選択は非常に生理的であるが、これらの方法は、他のTCRトランスジェニックモデルに適用することができます。我々はまた、遺伝子操作されたマウスに適用ポリクローナルレパートリーに正の選択を分析するための一般的な戦略を提示します。

生理的TCRトランスジェニックモデルとWTマウスでは、正の選択は、抗原遭遇の後、DP 鈍い段階に移る前に、DP 明るい段階から始まります。 DP胸腺細胞鈍いその後CD4SPまたはCD8SP胸腺細胞（ 図2B）になる前に、過渡的CD4 + CD8 loの段階に入る。成熟したSPの胸腺細胞は、高いTCR発現およびCD24（ 図2C）の損失によって特徴付け...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry

我々は、T細胞の発達を調べるためにフローサイトメトリーベースの方法を提示する<em生体内で&gt;</em&gt;野生型またはT細胞受容体トランスジェニック背景に遺伝子操作されたマウスを用いた。

フローサイトメトリーによる胸腺正と負の選択の検討

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

examination-thymic-positive-negative-selection-flow

Research

JoVE Journal

Immunology and Infection

21.6K ビュー.  University of Alberta. 我々は、T細胞の発達を調べるためにフローサイトメトリーベースの方法を提示する野生型またはT細胞受容体トランスジェニック背景に遺伝子操作されたマウスを用いた。

ビデオ: Examination of Thymic Positive and Negative Selection by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

フローサイトメトリーで固定し、生細胞におけるカルパイン活性の測定

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

免疫・感染学

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

フローサイトメトリーによる一次免疫細胞亜集団との蛍光ナノ粒子の相互作用を検出

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

フローサイトメトリーによるげっ歯類マラリア原虫のin vivo評価とメロゾイト侵略で

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

フローサイトメトリーによるラットの腎臓からのマクロファージの表現型の特徴

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

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 In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

離とマウスとヒトの肺胞マクロファージの体外培養

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血によって人間の Monocyte のサブセットの解析フローのフローサイトメトリー解析

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

7 免疫細胞サブセット 2 螢光色素の差別フローサイトメトリー

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

好中球系統細胞の質量細胞法解析に対する全骨髄の調製

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...