저자는 표면 형광 현미경에 관한 기술적 조언 박사 레오 니드 볼코프 에릭 보우 차드을 감사드립니다. 경제적 지원 GB의 건강 연구 (보조금 # 1 MOP-93781)의 캐나다 연구소에 의해 자금 지원

동물 보호는 셔 브룩 대학 동물 관리 및 사용위원회에 따라이었다. 1. 튜브 준비 <ol><li> 셀 소팅 전날, 5 ㎖ 폴리 프로필렌 둥근 바닥 튜브에 열 활성화 된 우 태아 혈청 (FBS) 1-2 mL를 넣고 15 mL 및 50 mL의 폴리 프로필렌 원추형 튜브에 관한 것이다. 중요한 단계 : 프로토콜에서 사용되는 모든 튜브가 코팅 된 것을 확인합니다. 참고 : FBS 코팅 튜브 벽에 달라 붙는 세균 세포를 방지 할 수 있습니다. </li><li> 천천히 튜브를 균일하게 회전을 사용하여 코트 4 ° C에서 반전에 의해 O / N을 섞는다. </li><li> 다음 날, 경사에 의해 코팅 된 튜브에서 FBS를 제거합니다. <ol><li> * 중요한 단계 : 5 ML의 폴리 프로필렌 둥근 바닥 튜브 및 셀 제조에 사용 (15)과 50 ㎖ 튜브 (프로토콜이 2.11에 2.1 단계) 완전히 패닝 (측면 스트림 즉, 부정확 편차)을 방지하기 위해 FBS의 비워되어 있는지 확인합니다. FBS를 제거하기 위해 P200 피펫을 사용합니다. <br/&gt; 참고 : 이전에 셀 또는 셀 정렬하는 동안 사용되는 다른 튜브의 오염의 손실을 초래할 수 정렬에 튜브에 잔류 FBS, </li><li> 라운드 5 ML의 폴리 프로필렌 정제 된 세포를 수집하는 데 사용 하단 튜브 FBS (약 100 ~ 200 μL)의 작은 잔류 볼륨을 남겨주세요. </li></ol></li></ol> 2. 휴대 준비 <ol><li> O를 CO 2 질식 이어 2 / 이소 플루 란 (당시 2 %로 유지 5 %에서 유도)를 사용하여 마취 한 남성 마우스를 희생. </li><li> 소비세 및 디 캡슐 모두 고환과 1.5 ML 튜브로 버퍼를 정렬 500 μL에 작은 가위로 말하다. </li><li> 그대로 100-1,000 μL 팁과 후 정액을 운반하는 최초의 잘린 100-1,000 μL 팁과 1.5 ML 튜브에 부드러운 상하 피펫으로 세관,과에서 세척 생식 세포. </li><li> 40 μm의 셀 스트레이너를 사용하여 하나의 여과 공정에 의해 파편과 덩어리를 제거하고 50 ML 원뿔 튜브 이전에 여과 액을 수집iously 1 단계에서 FBS로 코팅. </li><li> 이전에 1 단계에서 코팅 된 15 ML 원뿔 튜브에 총 3 ㎖의 볼륨 및 전송 여과까지 버퍼를 정렬 한 번 필터를 씻으십시오. </li><li> 0.8 mM의 EDTA의 최종 농도를 얻도록 50 mM의 EDTA 세포 현탁액의 pH를 8 밀리 리터당 (3 ㎖, 48 μL)의 16 μL를 추가한다. </li><li> 생식 세포를 해결하려면, 천천히 우유 정지를 만들어 부드러운 교반 10ml를 피펫 (낮은 속도로 소용돌이)를 사용하여 얼음처럼 차가운 100 % 에탄올 3 볼륨을 추가합니다. 중요한 단계 : 에탄올을 천천히 첨가 세포 제제의 무결성을 유지하는 것이 중요하고 중요하다. 우리는 빠른 에탄올 첨가 효율 정렬 주요한 감소의 결과로 세포 용해를 유발한다는 지적. 3 ml의 세포 현탁액의 경우, 에탄올의 9 ㎖를 약 1 분으로 첨가되어야한다. </li><li> 반전에 의해 가끔 혼합 15 분 동안 얼음에 세포를 품어. </li><li> 원심 분리기 세포 5 분 800 XG에 서스펜션과 맑은 제거상층 액. </li><li> 부드럽게 정렬 버퍼 2 ㎖에 고정 생식 세포를 재현 탁. </li><li> SYTO16 DNA 염료 (DMSO 1 mM의 용액)의 4 μl를 추가하고 (빛으로부터 보호) 적어도 30 분 동안 품어. 주 : 최상의 정렬 결과는 쉽게 정자의 고도로 압축 된 핵에 도입되는 DNA 투과성 염료이므로 SYTO16를 사용하여 얻어진다. </li></ol> 3. 세포 분류기는 설정 참고 : 여기, 4 레이저 (405 nm의 - 보라색, 488 nm의 - 블루, 561 nm의 - 노란색 - 녹색, 633 nm의 - 빨강) 20 매개 변수 BDFACSAria III 세포 분류기 사용됩니다. BD FACSDiva 6.1.3 소프트웨어는 데이터를 시각화 및 분석하는 데 사용된다. 설정이 사용 분류기의 종류에 따라 달라질 수있다. <ol><li> 유체 역학 종료 절차 (소프트웨어의 &quot;사이토&quot;메뉴) 분류 이전시 칼집 유체로 70 % 에탄올을 실행하여 FACS 분류기를 소독. 소프트웨어의 도구 모음에서 &quot;사이토&quot;를 선택하고 &quot;유체 쉬를 선택. &quot;를 utdown 프로세스에 의해 언급 된 단계를 수행합니다. </li><li> 제조 및 수정 및 오염물을 제거하기 위하여 0.22 μm의 필터로 1X PBS 필터. 탱크 내부의 상부에 용접선에 칼집 탱크를 채우기. </li><li> 소프트웨어를 시작하고 로그인합니다. 이전에 정렬에 적어도 30 분 동안 레이저를 따뜻하게 외과 분류기를 시작합니다. 유체 역학에서 에탄올을 세척하는 두 유체 시작 프로세스를 실행합니다. 소프트웨어의 도구 모음에서 &quot;사이토&quot;를 선택하고 &quot;유체 시작&quot;을 선택합니다. 프로세스에 의해 언급 된 단계를 수행합니다. 참고 :이 정상 칼집 유체 (1X PBS)와 유체 공학을 복원합니다. </li><li> 정렬 블록과 증류수로 편향 판을 청소하고 철저하게 그들을 건조. 초음파 욕에서 2 분 동안 증류수 튜브에 넣고 100 ㎛ 노즐을 초음파 처리. 철저하게 노즐을 닦습니다. </li><li> 흐름 셀에 100 μm의 노즐을 삽입하고 12 노즐 잠금 레버를 시계 방향으로 돌립니다시간 위치. 20 PSI에 칼집 압력을 설정합니다. </li><li> 스트림의 전원을 켜고 (약 30) 주파수에 대한 목표 값을 얻기 위해 진폭을 조정, 1 (150의 주위에) 삭제하고 (12 정도) 갭과 안정적인 방울 breakoff 패턴을 (몇 위성 방울) 설정합니다. 감쇠를 끕니다. </li><li> 스트림이 직선이고 정렬 블록의 각도를 변경하여 폐기물 흡입기의 중심 안타 있는지 확인합니다. 참고 : 10 PSI에서 130 μm의 노즐도 테스트하고 명백한 차이는 발견되지 않았다. </li><li> 노란색 - 녹색 레이저에 대한 파란색, 보라색의 적색 -77.39, 37.33 및 -39.85 0으로 설정 레이저 지연. </li><li> 노란색 - 녹색 레이저에 대한 파란색, 보라색의 붉은 1.0, 0.75 및 0.96에 대한 1.14로 확장 설정 지역. </li><li> (소프트웨어의 &quot;사이드 스트림&quot;창에서) 테스트 정렬을 사용하여, 그 측면 스트림 패닝되지 않습니다 있는지 확인하십시오. 그들은 middl 충돌되도록 쪽 &quot;스트림&quot;윈도우에서, 각 사이드 스트림의 전압을 조정 슬라이더테스트 수집 튜브의 전자. 중앙 스트림 꽉 없으면 중앙 스트림을 제한하는 2 번째, 3 번째, 4 번째 드롭 설정을 조정. </li><li> 소프트웨어 ( &quot;사이토&quot;메뉴)에서 사이토 설치 및 추적 (CS &amp; T) 응용 프로그램을 시작합니다. </li><li> 제조업체의 지침을 사용하여 세포 계수기의 성능 특성 및 추적을 자동화하기 위해 350 μL 당 1 방울의 세포 계측기 설치 및 추적 구슬을 사용합니다. </li><li> CS &amp; T 응용 프로그램을 종료합니다. CS &amp; T 설정을 적용, 스트림 매개 변수는 여전히 안정적인 방울 breakoff 패턴을 할 수 있는지 확인하고 ( &quot;Breakoff 창&quot;에서) 제조 업체의 지침에 따라 스위트 스폿 (Sweet Spot) 버튼을 켭니다. </li><li> 형광 비드를 사용하는 드롭 지연 값을 최적화 (자재 표 참조). <ol><li> 홀더에 수집 튜브를 설치합니다. 정렬 블록 문을 엽니 다. &quot;사이드 스트림&quot;창에서 &quot;를 선택 한 후 전압을 켭니다시험 종류 &quot;및 서랍을 열고 수집 튜브에 액세스 할 수 있도록 흡인기 서랍 버튼을 클릭합니다. </li><li> 그들이 관의 중심으로 이동되도록 사이드 스트림을 최적화한다. 필요에 따라 값과 슬라이더를 조정합니다. </li><li> 정렬 블록 문을 닫고 중앙의 밝은 구슬 자리를 얻기 위해 마이크로 미터 다이얼을 조절해야합니다. 선택 취소 폐기물 서랍, 시험 종류 및 전압. 컬렉션 튜브를 제거합니다. </li><li> 제조자의 지시에 따라 드롭 지연을 최적화. <ol><li> 간략하게, 비드 바이알을 격렬히 흔들 PBS 0.5 ㎖에 구슬 1 방울을 희석. </li><li> 제조업체의 지침을 사용하여 소프트웨어에서 사용할 수있는 드롭 지연 실험 템플릿을 사용하여 드롭 지연을 설정합니다. 또한, 드롭 지연을 설정하는 자동 드롭 지연 기능을 사용합니다. </li><li> 형광 비드 튜브를로드하고 임계 속도가 될 때까지 3,000 ~ 4,000 이벤트 / 초를 유량을 조정한다. 정렬 정밀도를 설정하려면 &quot;초기&quot;최종LY &quot;정렬 레이아웃&quot;창에서 &quot;미세 조정&quot;에. 광학 필터를 선택하고 구슬을 정렬합니다. 100 %가 왼쪽으로 분류되어 있습니다 ~까지 드롭 지연을 조정합니다. 참고 :이 단계가 있는지 확인하는 것이 중요하다 측 스트림에 관심 종류의 세포. 구슬과 광학 필터는 100 % 액적 편차 부근 달성하는 데 사용된다. </li></ol></li></ol></li><li> 금융위원회 검출기 블록의 왼쪽 끝에 FSC 1.5 ND (중성 농도) 필터를 놓습니다. 2.00 μS에 창을 확장하고 ( &quot;사이토&quot;창에서 &quot;레이저&quot;탭에서) 1.00 FSC 영역 확장을 설정합니다. </li><li> 샘플 튜브를 넣기 전에, 응집을 피하기 위해 샘플 라인의 끝에 50 ㎛의 샘플 라인 필터를 배치했다. 이렇게하려면, &quot;사이토&quot;메뉴에서 &quot;샘플 필터 변경&quot;을 선택합니다. </li><li> 사이드 스트림 사이에서 패닝이없이 양호한 분리를 달성하고 polypropyle 달라 붙는 것을 방지하기 위해 세포를 샘플 유체의 조성물을 시험NE 튜브. <ol><li> 샘플 튜브를로드 편향 판을 켜고 서랍이 닫힌 &quot;테스트 정렬&quot;을 선택합니다. 분리가 너무 많이 패닝없이 발생하는 경우 측면 스트림 봐. 주 : 패닝 인해 샘플 버퍼에 높은 단백질 농도 쉽다. </li></ol></li><li> 외과 악기로 샘플 튜브를 넣습니다. 300 rpm의 교반과 샘플 4 ° C의 온도 샘플을 선택한다. &quot;취득 대시 보드&quot;에서 &quot;데이터를 획득&quot;을 선택합니다. </li><li> 필요한 경우 (3.0의 최대 유량) / 초 5000 이벤트 최대 적절한 이벤트 레이트를 달성하기 위해 시료를 희석. 분류 세포의 순도를 유지하도록 엄격하게 이러한 한계를 존중한다. </li><li> 분석 및 세포를 정렬 관심 인구 간섭없이 이물질을 제거하기 FSC 임계 값을 최적화한다. 대수 눈금에서 만들기 위해 30분의 530 필터와 광 승산기 튜브 (PMT)의 검출 전압을 조정반드시 전체 인구의 형광 신호는 스케일에 들어가고, 그 양의 모집단의 게이트 (도 1, 게이트 1). </li><li> 앞으로 캐터 영역 (FSC-A) / 사이드 캐터 지역 SYTO16 긍정적 인 인구 (SSC-A) 플롯을 생산하고 약 110 V 선형 규모에 FSC-을 조정하고, 약 150 V로 SSC- 매우 큰 세포와 세포 집합체를 제외하는 동안 로그 스케일의 모든 이벤트를 시각화합니다. </li><li> &quot;정렬 레이아웃&quot;창에서 &quot;정렬 정밀 모드&quot;설정 &quot;4 방향 순도&quot;에 (수율 마스크 : 0, 순도 마스크 : 32, 위상 마스크 : 0). 연속 정렬에 대해 &quot;대상 이벤트&quot;메뉴에서 &quot;연속&quot;을 선택합니다. </li><li> ( &quot;정렬 레이아웃&quot;창에서) 튜브에 해당하는 필드에 정렬하는 인구를 추가합니다. 높은 중간 주파수와 끝에서 낮은 주파수를 가진 사람들과 장소의 인구. 정렬하는 동안 2500 (V)의 전압 판을 설정 샘플 모음 (T)를 유지얼음에 ubes. </li></ol> 4. 셀 정렬 <ol><li> 즉시 정렬 전에, 둥근​​ 바닥 튜브를 이전에 코팅을 5 ml 폴리 프로필렌 50 μm의 필터를 사용하여 필터 세포. </li><li> 버퍼를 정렬 1-2 ㎖로 광범위하게 필터를 씻으십시오. </li><li> 그림 1에 설명 된 게이트 방식 다음 488 nm의 레이저를 갖추고 세포 분류기를 사용하여 정렬 고정 생식 세포는. 이전에 얼음 섹션 1에 코팅을 5 ml 폴리 프로필렌 둥근 바닥 튜브의 정제 분수를 수집합니다. <ol><li> FBS의 100 ~ 200 μL가 튜브 벽에 정렬 된 세포의 부착을 방지하기 위해 수집 관에 보관되어 있는지 확인합니다. </li><li> SYTO16의 농도가 정렬 기간 동안 형광 변동을 방지하기 위해 포화되어 있는지 확인합니다. 정렬 그래픽 알렉사 488 형석 파라미터 (DNA 염색) 세포에서 염색 강도의 더 이상의 변화가 없을 때 SYTO16은 포화된다. 필요한 경우, 1 μl를 추가정렬 튜브 SYTO16은 포화에 도달한다. </li><li> 알렉사 플 루어 488 대 사건 번호 : 다음 매개 변수를 표시하는 그래픽의 총 이벤트를 표시합니다. </li><li> 도 1에 도시 된 바와 같이, 게이트 1 DNA 양성 염색 세포를 선택한다. <ol><li> 매개 변수로 FSC-대 SSC-를 사용하여 그래픽에 게이트 1 표시 셀 <ol><li> 그림 1에 켰을 때, 게이트 2 셀을 선택합니다. </li><li> 매개 변수로 알렉사 플 루어 488 대 FSC-A를 사용하여 그래픽에 게이트 2 표시 셀. </li><li> 그림 1에 켰을 때, 게이트 2 그래픽에 게이트 5 게이트 6 셀을 선택합니다. </li><li> 매개 변수로 알렉사 플 루어 488 대 488-W 알렉사 플 루어를 사용하여 그래픽에 별도로 게이트 5 게이트 6 표시합니다. </li><li> 선택 spermiogenesis는 게이트 (5) 그래픽에 1-9 정자 단계를 그림 1에 켰을 때, spermiogenesis는, 게이트 6 그래픽에 10-12 정자 단계를 반복합니다. </li></ol></li><li> 디스플레이매개 변수로 알렉사 플 루어 488 대 FSC-A를 사용하여 그래픽에 게이트 1에서 세포 <ol><li> 그림 1에 켰을 때, 문 3 문 4 셀을 선택합니다. </li><li> 매개 변수로 알렉사 플 루어 488 대 488-W 알렉사 플 루어를 사용하여 그래픽에 별도로 문 3 문 4 표시합니다. </li><li> 선택 spermiogenesis는 게이트 3 그래픽에 13 ~ 14 정자 단계를 그림 1에 켰을 때, spermiogenesis는 게이트 4 그래픽에 15 ~ 16 정자 단계를 반복합니다. </li></ol></li></ol></li></ol></li></ol>

<ol>
	<li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+production+of+functional+sperm+in+cultured+neonatal+mouse+testes.">In vitro production of functional sperm in cultured neonatal mouse testes.</a> Nature. 471 (7339), 504-507 (2011).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+production+of+fertile+sperm+from+murine+spermatogonial+stem+cell+lines.">In vitro production of fertile sperm from murine spermatogonial stem cell lines.</a> Nat. Commun. 2, 472 (2011).</li><li>Sun, X., Kovacs, T., Hu, Y. -. J., Yang, W. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+actin+and+myosin+during+spermatogenesis.">The role of actin and myosin during spermatogenesis.</a> Mol. Biol. Rep. 38 (6), 3993-4001 (2011).</li><li>Cheng, C. Y., Mruk, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+junction+dynamics+in+the+testis:+Sertoli-germ+cell+interactions+and+male+contraceptive+development.">Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development.</a> Physiol. Rev. 82, 825-874 (2002).</li><li>Laiho, A., Kotaja, N., Gyenesei, A., Sironen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+the+murine+testis+during+the+first+wave+of+spermatogenesis.">Transcriptome profiling of the murine testis during the first wave of spermatogenesis.</a> PLoS ONE. 8 (4), (2013).</li><li>Leduc, F., Maquennehan, V., Nkoma, G. B., Boissonneault, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+response+during+chromatin+remodeling+in+elongating+spermatids+of+mice.">DNA damage response during chromatin remodeling in elongating spermatids of mice.</a> Biol. Reprod. 78 (2), 324-332 (2008).</li><li>Meistrich, M. L., Mohapatra, B., Shirley, C. R., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+transition+nuclear+proteins+in+spermiogenesis.">Roles of transition nuclear proteins in spermiogenesis.</a> Chromosoma. 111 (8), 483-488 (2003).</li><li>Gr&#233;goire, M. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male-driven+de+novo+mutations+in+haploid+germ+cells.">Male-driven de novo mutations in haploid germ cells.</a> Mol. Hum. Reprod. 19 (8), 495-499 (2013).</li><li>Simard, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instability+of+trinucleotidic+repeats+during+chromatin+remodeling+in+spermatids.">Instability of trinucleotidic repeats during chromatin remodeling in spermatids.</a> Hum. Mutat. , (2014).</li><li>Wykes, S. M., Krawetz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+spermatogenic+cells+from+adult+transgenic+mouse+testes+using+unit-gravity+sedimentation.">Separation of spermatogenic cells from adult transgenic mouse testes using unit-gravity sedimentation.</a> Mol. Biotechnol. 25 (2), 131-138 (2003).</li><li>Yoshida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+round+of+mouse+spermatogenesis+is+a+distinctive+program+that+lacks+the+self-renewing+spermatogonia+stage.">The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage.</a> Development (Cambridge, England). 133 (8), 1495-1505 (2006).</li><li>Moreno, R. D., Lizama, C., Urz&#250;a, N., Vergara, S. P., Reyes, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+activation+throughout+the+first+wave+of+spermatogenesis+in+the+rat.">Caspase activation throughout the first wave of spermatogenesis in the rat.</a> Cell Tissue Res. 325 (3), 533-540 (2006).</li><li>Meistrich, M. L., Trostle-Weige, P. K., Van Beek, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+specific+stages+of+spermatids+from+vitamin+A-synchronized+rat+testes+for+assessment+of+nucleoprotein+changes+during+spermiogenesis.">Separation of specific stages of spermatids from vitamin A-synchronized rat testes for assessment of nucleoprotein changes during spermiogenesis.</a> Biol. Reprod. 51 (2), 334-344 (1994).</li><li>van Pelt, A. M., de Rooij, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+the+seminiferous+epithelium+after+vitamin+A+replacement+in+vitamin+A-deficient+mice.">Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice.</a> Biol. Reprod. 43, 363-367 (1990).</li><li>Getun, I. V., Torres, B., Bois, P. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+purification+of+mouse+meiotic+cells.">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. , (2011).</li><li>Kovtun, I. V., McMurray, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trinucleotide+expansion+in+haploid+germ+cells+by+gap+repair.">Trinucleotide expansion in haploid germ cells by gap repair.</a> Nature Genet. 27 (4), 407-411 (2001).</li><li>Bastos, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis.">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65 (1), 40-49 (2005).</li><li>Lassalle, B., Ziyyat, A., Testart, J., Finaz, C., Lef&#232;vre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+method+to+isolate+round+spermatids+from+mouse+testis.">Flow cytometric method to isolate round spermatids from mouse testis.</a> Hum. Reprod. 14 (2), 388-394 (1999).</li></ol>

The authors declare no competing financial interests.

정자 세포는 항상 정액을 운반하는 상피 세포의 복잡성뿐만 아니라 체외 문화의 제한된 성공을 주어 연구에 도전하고있다. 수년에 걸쳐, 많은 접근법은 다양한 종에서 생식 세포가 개발되었다 정제한다. 퍼콜 또는 소 혈청 알부민 구배로 중력을 이용하여 침전 정화 기법은 일반적으로 그대로 생식 세포의 우수한 수율을 제공하지만, 이러한 감수 배체 세포 및 정자 10 일부 세포 유?...

Haploid round spermatids differentiate into spermatozoa by a process called spermiogenesis. This involves many different steps including the acquisition of a flagellum, chromatin and cytoskeleton remodeling, condensation of the nucleus as well as the loss of most of the cytoplasm. These unique cellular events must be finely regulated in order to produce a mature functional gamete with an intact genome suitable for fertilization. Spermiogenesis can hardly be studied in vitro since no reliable cell culture system has so far been able to support progression through the different steps of the process. Moreover, actual in vitro techniques lead to a poor yield1,2. In vivo, proper transitions through the different steps of spermiogenesis are crucial for the natural functional integrity of the male gamete. Successful purification of spermatids according to their differentiation steps has never been accomplished with a level of enrichment sufficient to allow molecular characterization of spermiogenesis. For instance, purification of key steps of the spermatidal differentiation would be especially useful to study the developing acrosome, formation of the midpiece3, cell junction dynamics4, RNA dynamics5, chromatin remodeling process6,7 or genomic stability8. Purification of spermatids has been hampered by their progressive morphological transformation, the lack of known stage-specific external biomarkers, and their peculiar shape and size. 
Although most male germ cells display a direct relationship between DNA staining and ploidy (DNA content), we noticed that such positive correlation is no longer applicable to spermatids. This stems from our early observation that seminiferous tubule sections show variable intensity of DNA staining throughout the different spermiogenesis steps. Although DNA staining is consistent with their haploid set of chromosomes from spermiogenesis steps 1 to 7 (round spermatids), we observed a sharp increase in fluorescence intensity with DAPI or SYTO 16 around the onset of nuclear reorganization and chromatin remodeling (spermiogenesis step 8) reaching a peak at the onset of nuclear condensation (spermiogenesis steps 11-12). Following condensation of the nucleus, DNA staining intensity decreases until spermiation (spermiogenesis step 16). We surmised that this was likely associated with the formation of their peculiar chromatin structure transition where histones are replaced by protamines. We therefore developed a reliable flow cytometry method that allows the separation of spermatids using the variation of DNA intensity of spermatids as a main selection parameter.
A simple flow cytometry approach is described to separate mouse spermatids with high purity (95-100%) based on their apparent DNA content (SYTO16 staining), size and granulosity. Spermatids are separated into four populations; spermiogenesis steps 1-9, 10-12, 13-14 and 15-16. Purified spermatids are suitable for genetic/genomic analysis, as well as proteomic applications as described in a recent publication from our group9.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Isoflurane</td>
			<td style="width:119px;">ABBOT</td>
			<td style="width:129px;">05260-05</td>
			<td style="width:184px;">For mouse anesthesia before euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Fetal bovine serum</td>
			<td style="width:119px;">Wisent</td>
			<td style="width:129px;">90150</td>
			<td style="width:184px;">For tube coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">1X PBS</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">EDTA</td>
			<td style="width:119px;">BioShop</td>
			<td style="width:129px;">EDT</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">HEPES</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:129px;">H</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">100 % Ethanol</td>
			<td style="width:119px;">Les alcools de commerce</td>
			<td style="width:129px;">092-09-11N</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">SYTO 16</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:129px;">S7578</td>
			<td style="width:184px;">DNA staining</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">5 ml polypropylene round bottom tubes</td>
			<td style="width:119px;">BD Falcon</td>
			<td style="width:129px;">352063</td>
			<td style="width:184px;">Sorted cells collection</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">15 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">1500</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">50 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">5000</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">TEC4 anaesthetic vaporizer</td>
			<td style="width:119px;">Ohmeda</td>
			<td style="width:129px;">1160526</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">CO2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">C799117902</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">O2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">O254130501</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Homemade mouse gas chamber</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">40 &#181;m Falcon cell strainer</td>
			<td style="width:119px;">Corning Incorporated</td>
			<td style="width:129px;">352340</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">50-micron sample line filters</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">649049</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Vortex mixer</td>
			<td style="width:119px;">Labnet international, inc.</td>
			<td style="width:129px;">S0200</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Dynac centrifuge</td>
			<td style="width:119px;">Clay Adams</td>
			<td style="width:129px;">101</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Celltrics 50 &#181;m filters</td>
			<td style="width:119px;">Partec</td>
			<td style="width:129px;">04-004-2327</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">488 nm laser-euipped cell sorter</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">FACSAria III</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:135px;">Accudop Fluorescent Beads</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">345249</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">Sorting Buffer: 1X PBS, 1mM EDTA pH 8.0, 25mM HEPES pH 7.0, 1%FBS</td>
			<td></td>
			<td></td>
			<td style="width:184px;">FBS is heat-inactivated. Make fresh solution, 0.22 &#956;m filtered and keep at 4&#176;C.</td>
		</tr>
	</tbody>
</table>

step-specific sorting of mouse spermatids by flow cytometry

마우스 정자의 분화는 그대로 게놈과 수컷 생식 기능의 제조를위한 하나의 중요한 과정이 다음 세대로 전달 될 것이다. 지금까지, 이러한 형태 학적 변화의 분자 연구는 후속 분석을위한 spermatid 분화 이러한 중요한 단계의 적절한 분리를 가능하게하는 방법의 결여에 의해 방해되어왔다. 유동 세포 계측법을 사용하여 이러한 세포의 적절한 게이트에서 이전 시도 때문에 염색질 리모델링을받은 정자의 DNA 형광의 독특한 증가로 어려웠을 수있다. 이러한 관찰에 기초하여, 우리는 에탄올로 고정 마우스 정자 네 개체군의 재현성 정화를 허용 방식 계측법 단순 흐름의 세부 사항을 제공하는, 각각의 핵 리모델링 과정에서 다른 상태를 나타내는. 인구 농축 단계 특정 마커와 형태 criterions에를 사용하여 확인할 수있다. 정자 정제 게놈과 proteom 위해 사용될 수있다IC는 분석한다.

게이팅 전략 유동 세포 계측법에 사용 그림 1은 네 개의 순수 spermatid 인구를 정렬 유동 세포 계측법에 사용되는 게이팅 전략을 나타냅니다. 간단히, 긍정적 인 DNA 염색과 세포 (알렉사 플 루어 488-A)를 처음 크기 대 granulosity (SSC-A)를 보여주는 도트 플롯에 spermiogenesis 1. 정자가 12이 선택 단계들 게이트 (문 2)와 선택 ...

Watch this Scientific Journal Video about Step-specific Sorting of Mouse Spermatids by Flow Cytometry at JoVE.com

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

단계 별 마우스 정자의 정렬을 유동 세포 계측법에 의해

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

step-specific-sorting-of-mouse-spermatids-by-flow-cytometry

Research

JoVE Journal

Developmental Biology

10.4K 조회수.  Université de Sherbrooke. We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

비디오: Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Flow Cytometry에 의한 마우스 태아의 적혈구 전구 세포 (Erythromyeloid Progenitors)와 그 자손의 동정

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

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Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

분리 및 유동 세포 계측법에 의해 췌장 중간 엽 세포 분석

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

기계 조직 해리 및 유동 세포 계측법에 의한 포유 동물 수컷 세포의 다중 종 정제에 대한 표준화 된 접근법

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

조 혈 줄기 세포 이식 Myelodysplastic 증후군의 평가에 사용

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

칼슘 유입, 기 공 형성 및 성숙한 신경 조상 세포 수용 체 P2X7에 의해 식 균 작용을 조사 하기 위해 실시간 라이브 셀 Cytometry

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

질량 세포 측정에 의한 세포 단백질 발현의 세포 주기 특이적 분석을 위한 마우스 피부 각질 세포의 분리 및 염색

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

표준 실험실 장비를 사용하여 마우스 고환에서 파키텐 정자 세포와 정자의 농축

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

유동 세포형을 사용하여 개발 중인 제브라피시에서 변경된 혈액 형성을 감지하고 양량화합니다.

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

교란되지 않고 손상된 마우스 골격근에서 섬유 지방 형성 전구 세포 및 근육 줄기 세포의 FACS 분리 및 배양

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

유세포분석에 의한 마우스 조직으로부터 파열 형성 단위 및 콜로니 형성 단위 적혈구 전구체의 식별 및 분리

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...