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>
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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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.5K 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

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Electroporation에 의한 배아 마우스 달팽이의 Explants의 문화 유전자 전송

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

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In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

마우스 배아의 자궁 내 인트라 심장 토마토 렉틴 주사에서 신장 혈류를 측정하기 위해

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

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 ...

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, ...

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

자기 활성화 세포 정렬에 의해 분리 된 혈관 주위 지방 조직으로부터의 지방 전구 세포 전구 세포의 확장 및 지방 생성 유도

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

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. ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

마우스에서 초기 임신 특성에 대 한 3 차원 높은-주파수 초음파의 새로운 사용

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

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 ...