저자는 자신의 기술 지원을 빙 장을 감사드립니다. 이 작품은 건강 연구 (걸레-86595)에 대한 캐나다 연구소에 의해 자금을 지원했다. TAB는 CIHR 새로운 탐정과 AHFMR 학술 수 있습니다. 박사와 AIHS 풀 타임 학생이기 - QH는 CIHR 캐나다 대학원 장학금으로 지원됩니다. SAN은 퀸 엘리자베스 II 대학원 장학금으로 지원됩니다. 박사 - AYWS는 NSERC 대학원 장학금으로 지원된다.

실험 프로토콜의 전반적인 구조에 대해 1 그림을 참조하십시오. 1. 해부 <ol><li> 60 X 15mm 페트리 접시에 장소 무균 스틸 메쉬 화면. 하나의 단위 조직 샘플 당이 필요합니다. </li><li> 각 요리에 행크의 균형 소금 솔루션 (HBSS)의 5 ML을 추가합니다. 얼음 요리세요. </li><li> CO 2와 쥐를 안락사시켜야. </li><li> 분해 표면에 보안 마우스, 최대 직면하고 복부 쪽. 살균에 대한 70 %의 에탄올과 및 모피는 아래 헝클어되어 있는지 확인하는 스프레이 마우스. </li><li> 수술 가위를 사용, 그냥 성기 위의 복부 피부에 복부​​ 절개를하여 절개를 시작합니다. 턱에 절개 위쪽을 연장합니다. </li><li> 중간 선에서 모든 다리를 따라 절개를 확장합니다. 느슨한 피부를 당겨하고 분해 표면에 내려 핀. </li><li> thymus를 수확 : <ol><li> 절개을 할 수있는 흉골의 하단 끝을 들어 봐요.</li><li> 간을 피 갈비를 분리하고 각 측면에 갈비를 절감 가로막을 잘라. 폐와 심장을 피하기 위해 관리하고, 각 쪽의 흉곽 위쪽으로 자른다. </li><li> 흉곽을 다시 집게로 부드럽게 당긴다. thymus은 심장 위에있는 흰색 bilobed 기관이다. 엽 (叶)의 하단을 파악하기 위해 포셉의 평면 가장자리를 사용하여 부드럽게 thymus을 뽑아 메쉬 화면에 배치. </li></ol></li><li> 비장을 수확 : <ol><li> 비장은 간 아래에있는 마우스의 복강의 왼쪽에있는 빨간색 서핑 보드 모양의 기관이다. </li><li> 복강에 절개를합니다. 부드럽게 결합 조직을 멀리 감히 두 번째 쌍을 사용 포셉 한 쌍의와 함께 비장을 당겨. 별도의 메쉬 화면에서 비장를 놓습니다. </li></ol></li></ol> 2. 셀 준비 <ol><li> 3 ML의 주사기에서 플런저를 사용하여 분쇄만 결합 조직과 지방 유적까지 메쉬 화면에 기관. 요리의 HBSS 세 번으로 메쉬를 씻어. 각 조직 샘플에 대한 새로운 플런저를 사용하십시오. <ol><li> 조직을 homogenizing에 대한 다른 옵션은이 방법 대신 사용할 수 있습니다. </li></ol></li><li> hemocytometer를 사용하여 셀을 계산합니다. </li><li> 335에서 원심 분리하여 펠렛 세포는 4 ° C.에 5 분을 위해 XG </li><li> 실온에서 10 분을위한 ACK 용해 버퍼 500 μl (0.15 M NH 4 CIA 같으니, 10 MM KHC0 3, 0.1 MM 그렇단이 EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.2)에 Resuspend splenocytes. 20 Resuspend thymocytes는 x 10 6 세포 / FACS 버퍼에 ML (PBS, 1 % FCS, 0.02 % 나트륨 azide)과 얼음에 둔다. </li><li> HBSS의 5 ML을 추가하여 isotonicity에 splenocytes을 반환합니다. 펠렛의 20 원심 분리 및 resuspend로 splenocytes X 10 6 세포 / FACS 버퍼에 ML. 세포가 무균 상태로 유지해야하는 경우, 당신은 무균 RPMI + 10% FCS를 사용할 수 있습니다. </li></ol> 3.유동 세포 계측법에 얼룩 세포 이 프로토콜의 목적은 wildtype (WT)와 HY의 CD4 마우스를 사용하여 긍정적이고 부정적인 선택의 분석을위한 전략을 설명하는 것입니다. 유동 세포 계측법 실험 설계, 획득, 분석에 대한 일반적인 고려 사항의 경우, 우리는 퉁 외하여 우수 리뷰에 독자 참조하십시오. 11 <ol><li> 나누어지는 4 X 10 96 - 웰 플레이트의 각 잘에 유동 세포 계측법 샘플 당 6 thymocytes뿐만 아니라 보상 제어 당 1 × 10 6 WT splenocytes. 유전자 변형 마우스를 사용하는 경우, 당신은 실험에 컨트롤로 WT 마우스를 포함해야합니다. </li><li> 얼음에 10 분에 anti-CD16/32 (클론 2.4G2)로 세포를 잠복기가 FC 수용체를 차단합니다. </li><li> 5 분에 대해 4 ° C에서 335 XG에서 판을 봐. 뚜껑을 제거하고 한 번 판을 flicking하여 우물에서 액체를 풀다, 세면대에 얼굴을 아래로. FACS 버퍼 200 μl의 각 우물을 Resuspend. 세탁을 반복합니다. </li><li> 아래에 나열된뿐만 당 FACS 버퍼 200 μl에 희석를 기준으로하여 각 항체의 최적 농도를 사용하여 항체 칵테일을 준비합니다. 각 항체의 최적 농도는 긍정적이고 부정적인 인구의 가장 큰 분리를 제공하는 데 필요한 최저 농도로 정의됩니다. 특정 항체에 대한 부정적인 인구가없는 경우, isotype 항체가 포함되어야합니다. 원하는 경우도 당 총 볼륨이 (물론 당 100 μl로 등)를 줄일 수 있습니다. <ol><li> WT thymus : 안티 - TCRβ, 안티 CD4, 항 CD8, 항 CD69 또는 안티 CD5, 항 CD24 </li><li> thymus HY의 CD4 : 안티 - HY TCR (T3.70), 안티 - CD4, 항 CD8, 항 CD69 또는 안티 CD5, 항 CD24 CD69 ro와 thymocytes에서 CD69 하이 인구의 더 나은 분리를 얻으려면, 그것은 다음, biotinylated 방지 CD69 주요 항체를 사용하는 것이 좋습니다 보조 fluorochrome - 복합 streptavidin을 포함하는 얼룩. 우리는 UCD5 착색에 대한 SE 같은 전략. </li></ol></li><li> 어둠 속에서 얼음에 30 분을위한 FACS 버퍼의 항체 칵테일 200 μl로 세포를 배양. </li><li> 항체 칵테일 착색이 동시에, 하나의 fluorochrome - 복합 항체 각 보상 제어를 얼룩. 이상적으로, 보상 착색은 항체 칵테일에 사용되는 것과 같은 항체을 포함한다. 많은 항원이 assayed되고 나면 그러나, 각각의 항원에 대한 얼룩은 큰 실험에 대한 가능한하지 않을 수 있습니다. 따라서, 항 CD4 또는 안티 CD8 중에 얼룩은 높은 이들 항원의 표현, 또는 보상 비즈의 사용으로 인해 권장합니다. 어둠 속에서 얼음에 30 분을위한 FACS 버퍼에 청바지. 한 보상 제어 흠없는 둡니다. </li><li> FACS 버퍼로 두 번 세포를 씻으십시오. </li><li> FACS 버퍼와 FACS 튜브로 전송에 Resuspend 세포. </li><li> 흐름 cytometer에 샘플을 수집. FSC-A의 수집 및에 FSC-W 포함이중 어 차별에 대한 llow. </li></ol> 4. 유동 세포 계측법 데이터 분석 - 비 TCR 트렌스 제닉 마우스 우리는 유동 세포 계측법 데이터 분석을 위해 FlowJo를 사용합니다. 비 TCR 유전자 변형 마우스의 게이팅 전략에 대한 2A 그림을 참조하십시오. <ol><li> SSC의 &quot;림프구&quot;인구에 전자 게이트에서 FSC-A를 사용합니다. </li><li> &quot;림프구&quot;인구 내에서, 세포 doublets 및 aggregrates을 제외 할 전자 게이트 FSC-W 그냥 ... 인구에 FSC-W에 의해 FSC-A를 사용합니다. 이렇게하면 &quot;중항&quot;문입니다. </li><li> &quot;중항&quot;게이트, CD4의 줄거리 CD8의 이벤트를 사용합니다. DN, DP 지루, DP 밝은에 문을 그려, CD4 그림 2B에 묘사 된대로 + CD8 이오, CD4SP 및 CD8SP 인구. </li><li> CD4SP 및 CD8SP 문에서 CD24 플롯에 의해 TCRβ을 만듭니다. 그림 2C에 도시로 문을 끌기 위해 사분면의 게이팅 도구를 사용합니다. </li><li> NE를 만듭니다CD69의 TCRβ : &quot;중항&quot;게이트에서 w의 줄거리. 문 A, B, C, D 그림 2D에 도시.을 그립니다 </li><li> CD5에 의해 TCRβ : &quot;중항&quot;게이트에서 다른 음모를 만듭니다. 그림 2E에 묘사 된대로 문 I, II, III, IV을 그립니다.이 긍정적 인 선택을 검토의 또 다른 전략이다. </li><li> 문 I, II, III, IV, A, B, C, D (그림 2D, E)에 &#39;중항 &quot;아래에서 DN, DP 지루, DP 밝고 CD4의 정수, CD4SP 및 CD8SP 문을 적용합니다. </li><li> 귀하의 대상 인구에 도달 할 때까지 이후의 각 게이트의 주파수에 의해 오르간 cellularity를​​ 곱하여 특정 하위 집합에있는 셀의 개수를 계산할 수 있습니다. 예를 들어, TCRβ 하이 CD69-CD8SP의 thymocytes의 수는 thymus cellularity X % TCR bhi에 의해 결정됩니다 CD69-(일부 D) X %의 CD8SP. <ol><li> 계산은 이미 라이브와 죽은 세포가 그렇게 &#39;lymphoc을 포함하지 않는 고려귀하의 계산 yte &quot;게이트. </li></ol></li></ol> 5. 유동 세포 계측법 데이터의 분석 - TCR 트렌스 제닉 마우스 TCR 유전자 변형 마우스의 게이팅 전략에 대한 3A 그림을 참조하십시오. <ol><li> 단계 이전 섹션에서와 같이 4.1 및 4.2을 따르십시오. </li><li> &quot;중항&quot;게이트에서 T3.70의 SSC 계획 및 게이트에 의해 T3.70을 만들 + 세포 인구를 항원 특정 T 세포 (그림 3B) 분석 할 수 있습니다. 일부 환경에서는 비 항원 특정 (T3.70-) T 세포의 인구에이 인구를 비교할 관심이있을 것으로 생각됩니다. </li><li> 내 T3.70 + 인구, 추첨 DN, DP, CD4SP 및 CD8SP 문 (그림 3C). <ol><li> 거의 T3.70 + 세포가있을 것 같은 WT 제어 샘플에 대해 &quot;중항&quot;게이트에서이 문을 그리거나 T3.70의 문을 만듭니다. </li></ol></li><li> CD8SP 게이트에서을 만들CD24 플롯으로 T3.70. 네 인구 (그림 3 층)에 셀을 분할 할 사분면의 게이팅 도구를 사용합니다. </li><li> WT 마우스와 HY의 CD4 마우스의 T3.70 + DP 구획 (그림 4A)의 DP 구획에서 CD69 표현을 묘사 겹쳐 히스토그램을 생성합니다. CD5 표현 (그림 4B)을 검사 반복합니다. </li><li> 4.8에서와 같이 관심의 각 하위 집합에있는 셀의 절대 수를 계산합니다. </li></ol>

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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>

관심 없음 충돌이 선언 없습니다.

여기에 제시된 프로토콜은 비 TCR 유전자 변형과 TCR 유전자 변형 마우스의 양수와 음수 선택을 확인하는 데 사용할 수 있습니다. 이 프로토콜은 표면 항원의 착색에 대해 설명합니다. 분자 메커니즘의 추가 분석을 위해, 그것은 종종 세포 염색법을 수행 할 필요가 있습니다. 우리는 전사 인자 대부분의 세포 내 단백질과 BD Biosciences의 Foxp3의 얼룩 키트의 BD Biosciences Cytofix / Cytoperm 키트를 사용합니다...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> HyClone 행크의 균형 소금 솔루션 </td><td> 열 과학 </td><td> SH30030.02 </td><td></td></tr><tr><td> 금속 메쉬 화면 </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> 배양 접시 (60 X 15mm) </td><td> 피셔 과학 </td><td> 877221 </td><td></td></tr><tr><td> 주사기 (3 ML) </td><td> BD Biosciences </td><td> 309657 </td><td></td></tr><tr><td> 원추형 튜브 (15 ML) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> 현미경 </td><td> Zeiss - 프리모 스타 </td><td> 415500-00XX-000 </td><td></td></tr><tr><td> Hemocytometer </td><td> Hausser과학적인 </td><td> 3110 </td><td></td></tr><tr><td> 96 - 웰 플레이트 </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> 멀티 채널 피펫 </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> 태아 송아지 혈청 </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> 인산염은 염분 버퍼 </td><td> 피셔 과학 </td><td> SH3025802 </td><td></td></tr><tr><td> 나트륨 azide </td><td> IT 베이커 화학 (주) </td><td> V015-05 </td><td></td></tr><tr><td> FcR 차단 시약 </td><td></td><td></td><td> 복제 2.4G2 </td></tr><tr><td> 안티 - 마우스 HY TCR </td><td> eBioscience </td><td> XX-9930-YY의 * </td><td> 복제 T3.70 </td></tr><tr><td> 안티 - 마우스 CD4 </td><td> eBioscience </td><td> XX-0042-YY의 * </td><td> 복제 RM4-5 </td></tr><tr><td>nti 마우스 CD8α </td><td> eBioscience </td><td> XX-0081-YY의 * </td><td> 복제 53-6.7 </td></tr><tr><td> 안티 - 마우스 CD24 </td><td> eBioscience </td><td> XX-0242-YY의 * </td><td> 복제 M1/69 </td></tr><tr><td> 안티 - 마우스 TCRβ </td><td> eBioscience </td><td> XX-5961-YY의 * </td><td> 복제 H57-597 </td></tr><tr><td> 안티 - 마우스 CD69는 Biotinylated </td><td> eBioscience </td><td> 13-0691-YY * </td><td> 복제 H1.2F3 </td></tr><tr><td> 안티 - 마우스 CD5는 Biotinylated </td><td> eBioscience </td><td> 13-0051-YY * </td><td> 복제 53-7.3 </td></tr><tr><td> Streptavidin </td><td> eBioscience </td><td> XX-4217-YY의 * </td><td></td></tr><tr><td> 흐름 cytometer </td><td> BD Biosciences - FACS 캔토 </td><td> 338962 </td><td></td></tr><tr><td> FACS 튜브 </td><td> BD Biosciences </td><td> 352052 </td><td></td></tr><tr><td> 유동 세포 계측법 분석 소프트웨어 </td><td> TreeStar - Flowjo </td><td> FlowJo V7 / 9 </td><td></td></tr><tr><td> HyClone RPMI - 1640 미디어 </td><td> 열 과학 </td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> fluorochrome와 YY는 유리 병의 크기에 따라 다릅니다에 의해 * XX는 다릅니다. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

건강한 면역 체계가 자기 항원에 허용 남아있는 동안 T 세포가 외국 항원에 반응해야합니다. T 세포 수용체 (TCR) α와 β loci의 무작위 다시 정리하기는 자신과 외국에 모두 항원 특이성의 광대 한 다양성과 T 세포 레퍼토리를 생성합니다. thymus에서 개발 중에 레퍼토리의 선택은 안전하고 유용한 T 세포를 생성 중요합니다. thymic 선택에 결함이 면역과 면역 결핍 장애 1-4의 발전에 기여하고 있습니다. T 세포 progenitors은 CD4 또는 CD8 공동 수용체를 표현하지 않는 2 중 부정 (DN) thymocytes로 thymus를 입력합니다. αβTCR하고 두 공동 수용체의 표현은 &#39;더블 긍정적 (DP) 단계에서 발생합니다. 자기 펩타이드-MHC (pMHC) thymic 세포에서 제공과 αβTCR의 상호 작용은 DP의 thymocyte의 운명을 결정합니다. 높은 연관성 상호 작용은 부정적인 선택과 elimina로 연결자기 반응성 thymocytes의 기. CD4 또는 자기 MHC (5)에 의해 제시 외국 항원을 인식 할 수 CD8 하나의 긍정적 인 (SP) T 세포의 긍정적 인 선택과 개발에 낮은 친화 상호 작용 결과. 긍정적 인 선택은 성숙 T 세포의 생성을 관찰하여 polyclonal (wildtype) TCR의 레퍼토리와 마우스에서 공부 할 수 있습니다. 그러나, 작은 항원 특정 인구의 삭제를 포함 부정적인 선택의 연구에 적합하지 않습니다. 대부분의 모델 시스템은 부정적인 선택을 공부하지만, 생리 이벤트에게 6 요점을 되풀이하는 능력에 따라 다를하는 데 사용되었습니다. 외인성 항원의 행정 thymocytes 7-9의 비 특정 삭제 될 수 있습니다 예를 들어, thymocytes의 체외 자극에 깊은 선택에 관여 thymic 환경을 부족합니다. 현재 생체 부정적인 선택에서 공부에 가장 적합한 도구 transg을 표현할 마우스 아르자기 항원 내생에 enic TCR의 특정. 그러나 많은 고전 TCR 유전자 변형 모델은 조기 부정적인 선택의 결과로, DN 단계에서 유전자 변형 TCRα 체인의 조기 표현을 특징으로하고 있습니다. 우리 연구소는 10 wildtype 마우스에서 발생하는 부정적인 선택 SP 전환에 DP 동안 발생 할 수 있습니다 유전자 변형 HY TCRα이 조건 DP 단계에서 표현되는 HY의 CD4 모델을 개발했습니다. 여기, 우리는 HY의 CD4 마우스 모델에서 thymic 긍정적이고 부정적인 선택을 검토 할 수있는 흐름 세포 계측법 기반의 프로토콜을 설명합니다. HY의 CD4 마우스의 부정적인 선택이 매우 생리이지만,이 방법은 다른 TCR 유전자 변형 모델에도 적용 할 수 있습니다. 우리는 또한 유전자 조작 마우스에 적용 polyclonal 레퍼토리에 긍정적 인 선택을 분석하는 일반적인 전략을 제시합니다.

생리 TCR 유전자 변형 모델 및 WT 생쥐에서 긍정적 인 선택 항원 만남 후 DP 지루 무대로 이동하기 전에 DP 밝은 단계에서 시작됩니다. DP 무딘 thymocytes는 CD4SP 또는 CD8SP의 thymocytes (그림 2B)가되기 전에 과도기적 CD4 + CD8에게 이오 단계를 입력합니다. 성숙 SP의 thymocytes은 높은 TCR의 표현과 CD24 (그림 2C)의 손실을 특징으로하고 있습니다. ...

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

우리는 T 세포 개발을 검토 할 수있는 흐름 세포 계측법 기반 방법을 제시<em&gt; 생체 내</em&gt; wildtype 또는 T 세포 수용체 유전자 변형 배경에 유전자 조작 쥐를 사용합니다.

유동 세포 계측법에 의한 Thymic 긍정적이고 부정적인 선택의 시험

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 조회수.  University of Alberta. 우리는 T 세포 개발을 검토 할 수있는 흐름 세포 계측법 기반 방법을 제시 생체 내 wildtype 또는 T 세포 수용체 유전자 변형 배경에 유전자 조작 쥐를 사용합니다.

비디오: 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.

유동세포계측법로 고정하고 살아있는 세포에서 Calpain 활동을 측정

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

연구

면역학 및 감염병학

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.

사람의 체외에서</em면역 불균형의 초기 국가의 인정을위한 심사 도구를> 억제

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.

유동 세포 계측법에 의해 주요 면역 세포 모집단과 형광 나노 입자의 상호 작용의 검출

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.

유동 세포 계측법에 의해 쥐 말라리아 기생충의 생체 내 평가와 Merozoite 침공

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.

유동 세포 계측법에 의해 쥐의 신장에서 대 식세포의 표현형 특성

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.

마우스 골수에서 형질 수지상 세포의 정화를위한 형광 활성화 셀 정렬

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.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

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).

전체 혈액에 의해 인간 Monocyte 하위 집합의 Flow Cytometry 분석

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.

2 형광 색소에 의해 7 개의 면역 세포 하위 집합의 차별 Cytometry 흐름

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.

호중구 계보 세포의 질량 세포 분석 분석을위한 전체 골수의 준비

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 ...