1. 샘플의 준비 <ol><li> 세포 농도가 악기 (3,000 500,000 세포 / ML 타입 M 카세트를위한, 또는 3,000 2,500,000 세포 / 타입 S 카세트에 대한 ML)의 작동 범위 내에 있도록 ORFLO 희석제 또는 적절한 희석제와 세포 현탁액을 희석. </li><li> 1시 20분 (타입 M 카세트) 또는 1시 5분 희석 (타입 S 카세트)에 노 희석에 1시 5분의 희석은 대부분의 포유 동물 세포 라인에 권장되지만 적절한 희석은 세포 종류와 심는 밀도에 따라 달라집니다. 정확한 숫자는 필요한 볼륨은 약 75 μL입니다. </li></ol> 2. 실행 샘플 <ol><li> 전원 버튼을 누르고 홈 화면이 표시됩니다에 의해에 Moxi Z를 돌립니다. </li><li> 트레이를 아래로 눌러 Moxi Z, 피펫 75μL 샘플이 ... 화면이 표시됩니다에 카세트를 삽입합니다. </li><li> 카세트의 채움 포트 (파란색 타원형에 75 μL 시료를 피펫어느 카세트 끝에 따라 1 또는 2, 레이블) 악기에 삽입되었다. 이들은 2 시험에 사용할 수있는 일회용 카세트입니다. 참고 : 장소 끝이 잘 전면 입술 아래 잡은되도록 ~ 45 ° 각도로 잘 직접로드에 피펫 팁. 분배하면 카세트를 입력하도록 공기의 주머니를 일으킬 수로 진공이 유체의 분배에 대해 &quot;출세&quot;하지 않는 것이 중요합니다. 그것은 분배하는 동안 잘 넘는 액체 형태의 작은 구슬 (물론 항목의 크기)를 가지고하는 것이 좋습니다. </li><li> 대부분의 포유 동물 세포를 카운트 들어, 시작하는 데 어디서나 화면을 만지지. 매우 작은 입자 (4-8 μm의 유형 M 및 3-8 μm의 타입 S의 평균 직경의 평균 직경)를 카운트 들어, Moxi Z는 작은 입자 모드에서 실행할 수 있습니다. 참고 : 카세트 타입은 화면 상단의 검은색 막대에 표시됩니다. 이 모드에서는 Moxi Z는 기본값으로 2-10 μm의 직경에 규모를 설정하고 카운트 u를 수행작은 세포 검출을위한 최적화된 매개 변수를 노래. 여기 터치 테스트를 시작하고이 모드에서 실행하도록 소형 입자 모드 버튼 (블랙 스퀘어) 누르십시오. </li><li> Moxi Z는 테스트를 시작하며 세포 개수 결과는 약 8 초 (타입 M 카세트) 또는 15 초 (타입 S 카세트)에 완료됩니다. </li><li> 곡선 피팅 및 MVI 계산이 자동으로 시작하고 몇 초 추가로 필요합니다. 적당한 발작이 발견되면, 결과는 자동으로 화면에 표시됩니다. </li><li> 게이팅 기본 수집 모드를 확인하려면 곡선이 게이팅 모드로 전환하려면 카운트 버튼을 맞추기 누르십시오. </li><li> 이때 카세트 장치에서 제거할 수 있습니다. </li></ol> 3. 관리 데이터 <ol><li> AutoMode 스케일링이 꺼져있는 경우, 결과는 초기 곡선 맞춤 2-34 μm의 직경의 규모 (유형 M 카세트)를받을 2-26 μ에 대해 표시됩니다M (타입 S 카세트). Automode 스케일링가 켜져있다면, Moxi Z는 자동으로 모든 곡선 맞춤 데이터가 표시됩니다 확보하면서 곡선 맞춤 인구의 넓이에 가장 가까운 범위로 가로 축 (X 축)를 확장할 것입니다. </li><li> AutoMode 스케일링 수동으로 높은 해상도에서 결과를 표시, 해제면, 2-26 μm의 아이콘 (M 타입) 또는 2-18 μm의 아이콘 (타입 S)를 만져. 참고 : 카세트 타입은 화면의 왼쪽 상단에있는 파란색 상자에 표시됩니다. 커브 - 피팅 기능뿐만 아니라 시각적으로 데이터의 미묘한 차이를 구별하는 사용자의 능력을 모두 향상시킬 수있는 작은 세포 또는 입자를 계수 때 권장합니다. </li><li> 수평 해상도를 증가시 확장 버튼을 동적으로 사용할 수있는 X 축 스케일 범위 사이의 사이클링을 할 수 있도록 조정합니다 : 2-34, 2-26, 2-18 및 종류 M 카세트를위한 2-10 μm의, 또는 2 타입 S 카세트에 대한 -26, 2-18 및 2-10 μm의. 이 기능은 availa입니다즉시 셀 카운트를 따라 경. </li><li> 곡선 맞춤 또는 게이트디 지역 카운트 정보 (세포 / ml) 쇼핑은 히스토그램의 왼쪽 아래에 블랙 박스에 표시됩니다. 평균 셀 크기 정보는 히스토그램의 오른쪽 아래에 블랙 박스에 표시됩니다. </li><li> MVI 값이 히스토그램의 오른쪽 상단에 파란색 상자가 표시됩니다. 검사는 작은 입자 모드에서 실행 되었다면 MVI이 적용되지 않고 &quot;MVI = SPM&quot;로 표시됩니다. 세포의 농도가 MVI 운영 범위, &quot;범위 MVI = 부족&quot;의 메시지 이외의 경우 또한 표시됩니다. </li><li> 현재 히스토그램을 저장하려면 완료 아이콘을 만질. 이것은 XXX는 해당 시험 번호입니다 &quot;테스트 XXX&quot;의 이름으로 데이터를 저장합니다. </li><li> 수동으로 게이트 히스토그램을 위해, 곡선 맞추기 카운트 결과 버튼을 켜고 만질. 이것은 게이팅 기본 수집 모드를 만듭니다. 다음 회를 성문을 설정하거나 생략하고 각 파란색 게이팅 마커 밀어미세 게이트 조정을위한 e 왼쪽 및 오른쪽 화살표 (이들은 파란 게이팅 마커를 감동시 표시)은 마커 사이에있는 세포가 포함됩니다. 히스토그램 수직 스케일은 자동으로 곡선 맞춤 인구 진폭을 기반으로 확장됩니다. 이 수직 스케일을 조정하려면 히스토그램 영역에서 슬쩍가 화면 위나 아래로. </li><li> 곡선 맞춤 모드로 디스플레이를 반환하고 곡선 맞춤 모드는 기본 수집 모드를 만들기 위해 게이트 카운트 결과 단추를 만져. </li><li> 그런 다음 결과를 저장하고 홈 화면으로 돌아갑니다 완료 아이콘을 누르십시오. 결과를 삭제하려면 영구적으로 시험 결과를 삭제하려면 언제든지 삭제 아이콘을 누르십시오. </li></ol> 4. 이전 획득 데이터의 분석 <ol><li> 저장된 테스트를 열려면 홈 화면의 히스토그램 아이콘을 누르십시오. </li><li> 최대 9 저장된 histograms에 대한 아이콘이 화면에 표시됩니다.관심 시험에 적절한 아이콘을 누르거나 더 많은 테스트 결과를 볼 수있는 아이콘이 아래 페이지 위로 또는 페이지를 누르십시오. </li><li> 각각의 히스토그램은 그것이 구원받은 방법에 따라 곡선 맞추기 카운트 모드 또는 게이트 카운트 모드 중 하나에서 열립니다. 게이트 카운트 모드에서 게이팅 마커는 독립적으로 각각 블루 게이팅 마커를 밀어서 원하는대로 배치 할 수 있습니다. 자동 게이트는 원하는 정상에 감동하여. 게이트 카운트와 곡선 맞추기 카운트 모드를 토글 만져. </li><li> 히스토그램의 수직 스케일을 조정하기 위해 줌과 Unzoom 아이콘을 누르십시오. 수직 스케일은 또한 수직으로 아래로 표시 최대 (증가) 또는 (감소)에 손가락을 보내주시면하여 변경할 수 있습니다. </li><li> 영구적으로 시험 결과를 삭제하려면 삭제 아이콘을 누르십시오. </li><li> 에 완료 아이콘을 누르면테스트 결과를 닫고 홈 화면으로 돌아갑니다. (참고 : 확대된보기가 저장되지 않습니다.) </li></ol> 5. PC로 데이터를 전송 <ol><li> 데이터는 PC 또는 Mac에 Moxi Z USB 케이블을 연결해하여 Orflo의 Moxichart 응용 프로그램을 사용하여 PC로 또는 USB 전송을 ​​통해 전송할 수 있습니다. 블루투스 전송의 경우 (MoxiChart) 먼저 Moxi Z 단위의 홈 화면에 블루투스 아이콘을 눌러 장치의 블루투스 ID를 결정합니다. </li><li> 컴퓨터에서 MoxiChart 응용 프로그램을 열고 오른쪽 상단에있는 블루투스 아이콘을 클릭하십시오. 다음 Moxi Z의 블루투스 아이디에 해당하는 장치를 선택하고 업로드된 파일에 대한 대상 폴더를 선택합니다. </li><li> Moxichart이 자동으로 블루투스 연결을 통해 지정된 디렉토리에 모든 파일을 전송합니다. </li><li> 블루투스 (MoxiChart) 또는 USB로 전송된 파일은 analy 열리고 수그들은 포맷되기 때문에, MoxiChart 응용 프로그램과 함께 직접 제드 자형의 것 또는. csv 파일은, 그들은 직접 추가 / 사용자 정의 분석을 위해 Microsoft Excel 또는 다른 데이터 분석 프로그램에서 열 수 있습니다. </li></ol> 6. 대표 결과 Moxi Z의 정밀도 및 정확도를 평가하려면, HEK-293 세포, HELA 세포, CHO-K1, 효모, Jurkat E6-1 세포, PC12 세포 및 건의는 0-500,000 세포에서부터 농도와 비드 현탁액을 정밀 보정 / ML (타입 M 카세트)과 0-2-2.5e 6 셀 / ML (타입 S 카세트)을 계산하고 모든 포유 동물 세포를 미국 유형 문화 수집 (ATCC)에서 얻은되었다 보습 바로 앞에 달린 풀베는 날 Z2와 Moxi Z &#39;를 사용하여 비교했다 그리고 교양 등 이전에 7-11을 설명했다. 간단히, 세포는 RPMI 1640 (Jurkat E6-1, PC12), MEM (HEK-293, HELA) 셀 또는 F12K (CHO-K1) 전지의 핵심 매체와 75mm 두 조직 문화 flasks에 정지에서 배양해되었다. 모든 미디어로 보충되었다10% 태아 소 혈청과 100 U penicillin/100 μg의 스트렙토 마이신. Flasks는 5퍼센트 CO 2와 37 ° C에서 인큐베이터 유지되었다. 세포는 삼일마다 passaged되었다. 효모 세포는 (X5, C. albicans, 빈 13) 기증과 즉시 같이 제공된 사용되었습니다. 타입 M 카세트 데이터와 타입 S 카세트에 대한 ~ 2 2.5e 6 셀 / ML위한 ~ 500,000 세포 / ML의 초기 샘플의 농도는 보습 바로 앞에 달린 풀베는 날 Z2를 사용하여 설립되었다. 후속 이론 농도 수준은 생리적 버퍼 (Isoton II 또는 행크의 균형 식염수)와 시리얼 dilutions을 통해 준비했습니다. 각각의 농도에서 동일한 샘플은 두 시스템에 의해 측정되었으며 서로 역모를 꾸몄다. 그림 2에서 볼, 강한 선형 상관 관계가 있었다 (타입 M R 2&gt; 0.9886, 유형 S R 2&gt; 0.9781) 모든 샘플에 대한 카운트 사이. 농도는 또한 Moxi Z와 Z2 모두와 이론 / 예상 세포 농도와 비교되었다 ( <str긴 사연&gt; 그림 3)을 다시 보여준 강한 선형 상관 관계 (타입 M R 2&gt; 0.9758, 유형 S R 2&gt; 0.9774). 정밀도를 평가하려면, HEK-293 및 HELA 세포 카운트는 보습 바로 앞에 달린 풀베는 날 Z2, Moxi Z 및 hemocytometer를 사용하여 수행되었다. 이력서는 200,000-300,000 셀 / ML 범위 5 별도 카운트에서 측정한 것으로 Moxi Z (타입 M 카세트), 보습 바로 앞에 달린 풀베는 날 Z2과 hemocytometer를 사용하여 셀 카운트의 편차의 평균 계수는 (이력서, N = 19) 계산되었다. Moxi Z의 작은 평균 이력서 (그림 4) Z2의 비교 및 보습 바로 앞에 달린 풀베는 날 기반의 계산 기술에서만 달성 가능한 정밀도의 높은 수준의 대표이다. Moxi Z 악기의 치수 정밀도는 구입, 정밀 보정 폴리스티렌 구슬의 측정을 통해 다음 평가되었다. Moxi Z에서 평균 입자 크기 측정 (n은 = 5)를 제조보고 크기가 역모를 꾸몄다되었다. 볼 수 있듯이 전N 그림 5는 Moxi Z 측정은 지속적으로 높은 선형 상관 관계 (R 2 = 0.9989)와 제조 업체 값을 일치. 크기와 카운트 정보 이외에도, Moxi Z는보고 Moxi의 생존 능력 지수 (MVI) 값을 가진 포유 동물 세포 배양 건강 시약이없는, 일반적인 평가를 제공합니다. 이 인덱스는 전체 미립자 카운트와 관련하여 관심 인구 세포의 수를 비율로 생성됩니다. MVI 측정 기능을 발휘하려면, MVI의 판독 (타입 M 카세트)가 표준 설명서 및 흐름 cytometric 죽은 / 실시간 측정 기법을 비교했다. 죽음 (&lt;10% 가능한) HEK-293과 HELA 세포의 인구는 37 희석제 (Isoton II, Beckman 보습 바로 앞에 달린 풀베는 날)을 포함한 영양소 프리, 나트륨 불화물 ° C로 살아있는 세포의 하룻밤 incubations을 통해 만들어진 제어 이론 생존 수준은 살아있는 죽은 세포의 ratiometrically 혼합 알려진 농도에 의해 준비되었다. 해상도ulting 솔루션은 hemocytometer 카운트 및 유동 cytometer 측정 모두에서 중복의 생존 능력 수준에 대해 분석했다. Hemocytometer 측정은 0.1 % Trypan 블루 솔루션 및 세포 전통 50분의 50 혼합되어 사용되었다. 유동세포계측법 측정은 Viacount 시약 및 제조 지정된 프로토콜 (머크)를 사용 구아바 PCA cytometer (머크)를 사용되었다. 마찬가지로 그림 6과 같이 HEK 세포와 HELA 세포 &quot;에 대한 유사 hemocytometer의 생존 비율로 MVI 데이터의 선형 적합의&quot;피어슨 상관 계수 값 (R 2) = 0.9937 및 R 2 = 0.9728, 각각 R이 있었다. 이러한 결과는이 접근법은 유동 cytometer 또는 hemocytometer를 사용하여 얻은 라이브 / 죽은 측정에 유리하게 비교할 세포 viabilities 광범 걸쳐 문화 건강 정보를 제공하는 것을 보여줍니다. 표 및 그림 (Dittami 외 2011에서.) 15 : 1 그림. 박막 보습 바로 앞에 달린 풀베는 날의 계산 : 전기 전류는 박막 카세트의 셀 감지 구역을 통해 전달됩니다. 세포 CSZ를 통해 하나의 파일을 흘러로서, 전압의 순간적인 증가는 Moxi Z &#39;로 측정된다 <img alt="그림 2" src="/files/ftp_upload/3842/3842fig2.jpg" /> 그림 2. Moxi Z 카운트는 보습 바로 앞에 달린 풀베는 날 Z2 카운트 비교할 수 있습니다. 세포 및 비드 현탁액의 동일한 카운트가 보습 바로 앞에 달린 풀베는 날 Z2와 Moxi Z &#39;를 사용하여 계산되었다해도 Moxi Z 카운트의 카운트 값 (N = 3-4)를 평균에 대하여 꾸몄다되었습니다 보습 바로 앞에 달린 풀베는 날 해당 Z2 건의 및 선형 끼워 맞춤 R 2 값이 결정됩니다. ) 종류 M 카세트 - 0의 농도 범위 - 500,000 셀 / ML. B) 타입 S 카세트 - 0의 농도 범위 - 2-2.5e 6 / ml.Error 바에 따르면 ± 1 표준 편차. 그림 3. Moxi Z 농도 측정은 이론적으로 세포 농도를 VS. ) 종류 M 카세트 - 이론 농도 값은 초기 500,000 세포 / ML 시료의 시리얼 dilutions에서 준비가되어있다 B) 타입 S 카세트 -. 2.5e 6 셀 / ML 시료 - 이론 농도 값은 초기 2의 시리얼 dilutions에서 준비되었다. 오류 바 ± 1 표준 편차를 나타냅니다. <img alt="그림 4" src="/files/ftp_upload/3842/3842fig4.jpg" /> 4 그림. 보습 바로 앞에 달린 풀베는 날 Z2, Moxi Z (타입 M 카세트) 및 Hemocytometer위한 변동 계수는. 각 접근 방식 HEK-293 및 HELA 세포 카운트의 편차의 평균 계수는 (이력서, N = 19) 별도 5에서 측정한 CVS로 계산되었다 300,000 셀 / ML 범위 - 200,000의 HEK-293과 HELA 셀 건의. 오류 바 ± 1 표준 편차를 나타냅니다. 그림 5. Moxi Z-Moxi Z를 사용하여 입자 크기 측정의 정밀도는 구슬 크기를 측정 (타입 M 카세트) 대 제조 업체 비드 크기를보고했다. 오류 바 ± 1 표준 편차를 나타냅니다. <img alt="그림 6" src="/files/ftp_upload/3842/3842fig6.jpg" /> 6 그림. MVI를 사용하여 문화 건강 평가 - hemocytometer를 사용하여 시각적, trypan-푸른 스테인드 생존 카운트 대 MVI 측정 (타입 M 카세트)와 구아바 PCA Viacount의 생존 능력의 비교.

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORFLO+Moxi+Z:+Revolutionizing+electronic+cell+count+accuracy+and+cell+viability+estimates+through+curve+fitting.">ORFLO Moxi Z: Revolutionizing electronic cell count accuracy and cell viability estimates through curve fitting.</a> Life Science Magazine, VWR International. , (2011).</li></ol>

그레고리 M. Dittami 그분 Ayliffe는 Orflo 기술, 설계, 제조 및 판매 Moxi Z, 리처드 D. Rabbitt이 Orflo 테크놀러지에게 지급 컨설팅 전문, 아르바이트를 제공하는 회사의 직원입니다. 이 문서에 대한 생산 및 무료 액세스가 Orflo 테크놀로지의 후원됩니다.

기본 구현은 보습 바로 앞에 달린 풀베는 날의 접근 방식은 측정의 전반적인 정확도 높은 결정적이 될 수 있습니다. 임계 영역은 둘 이상의 세포가 동시에 조리개를 통과하고 한 전기 사건으로 측정되는 &quot;우연 사건&quot;에 대한 원시 세포 카운트의 수정입니다. &quot;우연 사건&quot;은 세포 크기와 클러스터링의 정도 (데이비스 외 1967) 6를 포함한 여러 가지 요인에 따라 달라집니다 오류에 기여한다. 그 결과, 주어진 샘플에 대한 필수 우연 보정 널리 셀 / 입자 타입 간의 심지어는 동일한 세포 유형의 서로 다른 문화 사이의 변화, 예측 할 수 없습니다. 보습 바로 앞에 달린 풀베는 날에 의해 초기 출판물에 뿌리 이론을 설립 관찰 행사와 경험적으로 정해진, 입자 고유의 보정 계수, Z의 숫자에 따라 원료 카운트의 대수 조정을 적용하는 것입니다. 정확한 카운트가 제대로 t 스위치와 함께 얻을 수 있지만그의 보정 알고리즘은, 그것은 세포 종류와 정확히 그 값을 예측에 대응하는 어려움 사이의 Z 값의 큰 변화로 인해 그 실질적인 적용에 제한됩니다. 여기서 우연 보정 알고리즘과 함께 Moxi Z 커브 피팅으로 식별 &#39;참&#39;셀 카운트를 사용하여 Moxi Z는 동적으로 진정한 농도를 달성하기위한 우연의 일치를 위해 해결합니다. 500,000 세포 / ML (타입 M 카세트)와 3,000 - - 2-2.5e 6 셀 / ML (타입 S 카세트) 하이 엔드 보습 바로 앞에 달린 풀베는 날 Z2를 사용하여 얻은 결과를 비교하면, Moxi Z는 0의 농도 범위에 걸쳐 비교 카운트를 생산 . 또한 Moxi Z는 카운트에서 변화의 유사한 낮은 계수와 황금 표준 세포 계수의 보습 바로 앞에 달린 풀베는 날 - 기법의 특징은 입자 사이징의 정밀도로이 카운트 정확성을 실현. 또한, 여기에 제시된 데이터는 Moxi Z &#39;가 유용한 정보를 제공할 수 있음을 나타냅니다셀 문화의 전반적인 건강에 관한. Moxi Z는이 문화 건강 평가를 결정하는 독점적인 소프트웨어 알고리즘과 커브 피팅 접근 방식을 활용합니다. 분리 파괴 등 blebbing 같은 세포 죽음과 관련된 형태학의 변화, 그리고 용적 왜곡 (카타 오카와 Tsuruo 1996, Liegler 외 1995, 쉐리 던 외 1981) 12-14 불가능하게 Moxi-Z &#39;가 사이 impedimetric 차이를 구별하기 위해 만들어 인구 및 죽은 세포 / 파편 인구를 살고 있습니다. MVI는 입자 파편의 상대적인 기여도, 수축된 괴사성 세포, 그리고 관심있는 커브-장착되어 세포 인구를 식별할 수 있도록 문화 / 입자 크기 분포를 분석하여 생성됩니다. MVI 값은함으로써 전반적인 입자 인구 프로파일에 관하여 monodisperse 인구 카운트 인구 지수 또는 ratiometric 법안을 반영합니다. 이 값은 다음 algorithmically - 조정 기반의 인구 통계 및 경험적 관찰이다. Becaus전자 MVI 전통 염색법 접근 방식이 아닌 다른 매개 변수를 바라보고, 그것은 이러한 기술을 반영할 것으로 예상되지 않는 대신 세포 배양의 건강에 중요한 대안보기는 특히 파편과 미생물 오염 물질과 관련하여 제공합니다. 요약에서는 Moxi-Z 셀 카운터는 특허 박막 센서 기술과 결합 보습 바로 앞에 달린 풀베는 날 원리와 커브 - 피팅 소프트웨어 알고리즘을 사용하여 정밀 재현성 셀 개수의 assays, 셀 크기, 셀 건​​강을 제공하는 울트라 소형 benchtop 악기입니다 . 8초에 - 직경 25 미크론 (34 미크론 2의 다이나믹 레인지 -) 종류 M 카세트와 함께 4에서부터 입자를 계산이 가능합니다. 15 초 안에 - 직경 20 미크론 (26 미크론 2의 다이나믹 레인지 -) 유형 S 카세트와 함께 3에서부터 입자를 계산이 가능합니다. 또한, Moxi-Z는 시약의 사용없이 건강 평가를 수행합니다. 그것은 훨씬 더 적은 노동 inte입니다nsive 및 수동 계산보다는 주관적. 표준 보습 바로 앞에 달린 풀베는 날의 계수에 비해, Moxi가 훨씬 작습 빠르고, 더 많은 정보를 제공 상당히 적은 유지 보수가 필요, 사용하기 쉽게, 그리고 비용의 분수입니다.

<table border="1" ><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> ORFLO 희석제 </td><td> ORFLO </td><td> MXA006 </td><td></td></tr><tr><td> 카세트 디스펜서 </td><td> ORFLO </td><td></td><td> 편리한 분배 25 카세트 최대 쇼핑몰 </td></tr><tr><td> USB 케이블 </td><td> ORFLO </td><td></td><td> PC / Mac 또는 전원 어댑터에 악기를 연결 </td></tr><tr><td> 전원 어댑터 (미국 및 유럽 모델에만 해당) </td><td> ORFLO </td><td></td><td> AC 콘센트에 USB 케이블을 연결 </td></tr><tr><td> USB 플래시 드라이브 </td><td> ORFLO </td><td></td><td> 상점 Mox난 Z 소프트웨어 및 사용자 설명서 </td></tr><tr><td> 교정 확인 비즈 </td><td> ORFLO </td><td></td><td></td></tr><tr><td> 전자 보정 카세트 </td><td> ORFLO </td><td></td><td> 적절한 시스템 운영 및 교정를 확인하는 전자 카세트 </td></tr></tbody></table>

determination of mammalian cell counts, cell size and cell health using the moxi z mini automated cell counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

Watch this Scientific Journal Video about Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter at JoVE.com

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Moxi Z 초소형 자동 세포 계수기는 특허 박막 센서 기술과 사이징을 수행하는 독점적인 소프트웨어 알고리즘과 입자의 넓은 크기의 범위를 계산뿐만 아니라 monodisperse의 전반적인 건강을 결정하는 보습 바로 앞에 달린 풀베는 날 원리를 결합한 소설 악기 포유 동물 세포 배양. 이 프로토콜은 세포 배양의 건강을 계산하고 평가하는이 악기의 사용을 설명합니다.

Moxi Z 미니 자동 셀 카운터를 사용하여 포유류 세포 계산, 세포 크기와 세포 건강의 결정

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

determination-mammalian-cell-counts-cell-size-cell-health-using-moxi

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

24.6K 조회수.  Orflo Technologies. Moxi Z 초소형 자동 세포 계수기는 특허 박막 센서 기술과 사이징을 수행하는 독점적인 소프트웨어 알고리즘과 입자의 넓은 크기의 범위를 계산뿐만 아니라 monodisperse의 전반적인 건강을 결정하는 보습 바로 앞에 달린 풀베는 날 원리를 결합한 소설 악기 포유 동물 세포 배양. 이 프로토콜은 세포 배양의 건강을 계산하고 평가하는이 악기의 사용을 설명합니다.

비디오: Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

와이드 필드 현미경을 사용하여 장기 이미징 포유 동물 세포

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생물학

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

포유 동물 세포에 Glycoproteins의 N - 연결 설탕 체인 펄스 체이스 분석

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

샙터 셀 카운터를 사용하여 세포 배양 동안 휴대폰 번호를 결정

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

포유 동물 세포의 세포주기의 위치 분석

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

포유 동물 세포에서 헴 합성 레벨 측정

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

플라스미드 벡터를 생성 깁슨 어셈블리를 사용하여 인간의 신장 요인-1α의 발기인의 규정에 따라 FLAG-태그 단백질을 표현

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

세포 투과성의 Cys를 사용하여 포유 동물 세포에 직접 단백질 배달<sub&gt; 2</sub&gt; - 그의<sub&gt; 2</sub&gt; 아연 손가락 도메인

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Zebrafish 독도에서 단일 셀 해상도 칼슘 이미징 사용 하 여 베타 세포 기능 분석

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

혐 기성 성장 및 포유류 세포의 유지 보수

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...