1. 셀 카운팅의 중요성 <ol><li> 카운팅 세포는 체외 세포 문화에 대한 필요하지만 지루한 단계입니다. </li><li> 일관된 세포 농도는 실험 재현성과 정확성을 보장합니다. </li><li> 셀 카운트는 후속 실험, transfection 또는 감염 세포를 심는 불멸화 또는 변환을 평가, 세포 건강과 확산 속도를 모니터링하고, 셀 기반의 assays를위한 준비 중요합니다. </li><li> 에도 불구하고 셀 카운팅의 속도 및 정확도의 필요성, hemocytometer를 사용하여 카운트 세포를 조사 400 연구자의 71 %. hemocytometry 저렴한지만, 그것은 힘드는과 부정 확한 계산 결과를 사용자의 바이어스 및 남용에 적용됩니다. </li><li> hemocytometry에서 오류 소스는 다음과 같습니다 : 예제에서 고르지 세포 배포 예제에서 너무 많은 혹은 너무 적은 세포, 특정 세포가 정의된 계산 영역에 속하는 것인 지에 대한 주관적인 의사 결정, hemocytometer의 오염, 사용자에게 사용자의 변화를 hemocytometer 충전 속도, 그리고 변화. </li><li> 수동 계산과 관련된 지겨움을 완화하기 위해, 연구자의 29 %가 자동으로 세포 계수 장치를 사용하여 셀을 계산합니다. 그러나 대부분의 연구원이 큰 벤치 - 최고의 악기와 관련된 가격은 그들이 사용하는 장벽이다. </li><li> 샙터 전지 카운터는 상대적으로 낮은 비용으로 자동화 및 쿨터 계산의 정확도를 제공하는 자동화된 휴대용 장치입니다. </li><li> 이 시스템은 감지, 신호 처리, 데이터 스토리지 및 그래픽 디스플레이를위한 아날로그 및 디지털 하드웨어의 조합을 사용하여 소형 형식으로 임피던스 기반의 입자 검출의 쿨터 원칙을 고용합니다. </li><li> 일회용 센서 셀 크기와 서브 마이크론과 하위 picoliter 해상도에서 셀 볼륨에 의해 차별을 가능하게 microfabricated, 셀 감지 영역에 설계되었습니다. </li><li> 정밀 액체 처리 채널과 전자와 향상된, 샙터 전지 카운터 그래픽 히스토그램으로 세포 인구 통계를 표시합니다. </li></ol> 2. 카운팅을위한 단일 셀 서스펜션의 준비 <ol><li> 샙터 셀 카운터를 사용하려면, 세포의 단일 세포 현탁액을 diluting로 시작, 인산의 생리 버퍼. ML 당 10,000-500,000 세포의 농도가 최적입니다. </li><li> 1.5 ML의 microfuge 관에 희석 세포 100 μL을 전송합니다. </li></ol> 3. 샙터를 사용하여 셀 카운트 공연 <ol><li> 악기의 뒷면에있는 토글 키를 누른 후 누른 샙터 셀 카운터 켜고 나타나는 화면의 지시에 나올 때까지 기다리는 것이 좋습니다. </li><li> 메시지가 나타나면 악기의 앞쪽으로 향하게 전극 감지 패널로 샙터 단위의 끝에 센서를 부착합니다. </li><li> 샙터 센서 전자 감지 영역 및 통합 셀 감지 전극과 정밀 성형 샘플링 챔버로 구성되어 있습니다. 센서는 가능한 서브 마이크론 해상도 셀 크기를 구별하고, 하위 picoliter 해상도 휴대폰 볼륨을합니다. </li><li> 일관된 센서 공학, 동일한 샘플 볼륨이 계산되며 정확한 크기 측정이 만들어진 것을 보장합니다. </li><li> 일단 센서는 다음과 같이 화면의 지시에 따라 계산 프로세스의 각 단계는 수행 첨부 : </li><li> 화면이 읽을 때 &quot;시작 플런저를 잡아,&quot;플런저를 우울. 화면은 다음 &quot;잠수함 센서를.&quot;읽습니다 세포 현탁액 솔루션으로 센서 및 센서에 세포 현탁액을 대기음하는 플런저를 풀어 잠수함. </li><li> 플런저는 전기 신호로 작동하며 센서에 샘플 플러스 죽은 볼륨을 이끌어낼 수 있습니다. 그것이 pipetting과 마찬가지로 폭락의 속도 또는 정확도가 중요하지 않습니다. 이 과정은 몇 초 정도 걸립니다. 준비가되면 악기가 삐 것이며, &quot;로드 샘플&quot;이 화면에 표시됩니다. </li><li> 일단 샘플이가 microchannels를 통해 다음, 세포 크기와 세포 볼륨을 평가하기 위해 감지 영역을 통해 그림이 그려진 센서에로드됩니다. 샘플 50 μL가 계산되면 센서가 카운팅 막을 수있는 계기를 알려줍니다. </li><li> 계산이 완료되면, 악기의 화면은 &quot;완료 세어보세요. 센서를 제거하고 삭제하십시오.&quot;읽습니다 프롬프트를 따라하고, 센서를 제거하고 폐기. </li><li> 악기는 다음의 화면에서 셀 크기 또는 직경의 히스토그램뿐만 아니라 ML 당 세포 농도를 표시합니다. </li></ol> 4. 데이터 분석 <ol><li> 계산이 완료되고 히스토그램이 악기에 표시되면, 토글 버튼을 눌러서 게이트를 적용합니다. &quot;자동 게이팅&quot;또는 자동으로 히스토그램 프로파일을 기반으로 게이트를 집합으로 &quot;마지막 사용&quot;또는 이전 계산의 성문을 사용을 선택합니다. </li><li> 에 관계없이 선택의 문을 수동으로 미세 조정 데이터를 이동할 수 있습니다. 왼쪽 문을 선택하고 스크롤 다시 전환 버튼을 눌러원하는대로 게이트를 이동 ggle. </li><li> 그런 다음 오른쪽 게이트를 활성화하고 올바른 게이트를 배치 전환을 스크롤 다시 전환을 누르십시오. 다시 클릭하여, 새로운 세포 농도, 평균 세포 부피와 직경이 표시됩니다. </li><li> 토글도 날짜 사이의 디스플레이를 전환 양쪽으로 이동할 수, 시간, 셀 크기를 의미하고, 세포 볼륨을 의미합니다. </li><li> 최대 72 histograms에는 악기 자체에 저장할 수 있습니다. 개인 컴퓨터에 데이터를 업로드하려면 디스플레이 화면의 USB 케이블 포트를 통해 악기를 연결합니다. </li><li> PC에 두 번 &quot;연결&quot;을 누른 다음 소프트웨어를 실행하려면 샙터 소프트웨어 응용 프로그램을 클릭합니다. 모든 계산이 자동으로 소프트웨어에 포트를합니다. </li><li> 샙터 어플 리케이션 소프트웨어는 볼륨이나 직경 중 하나의 문화의 인구 분포를 상세하게 히스토그램을 표시합니다. 다운로드한 파일은 원시 데이터가 지정된 셀 또는 셀 지름 볼륨의 용기에 표시됩니다 Microsoft Excel을로 내보낼 수 있습니다. </li></ol> 5. 대표 결과 <ol><li> 나인틴 다른 세포 라인 카운트 위해 준비되었고 이론적 시작 농도는 쿨터 카운터를 사용하여 결정됩니다. 이러한 세포 suspensions 그러면 더욱 이론적 세포 농도에서 세포 suspensions를 얻을 희석되었다. </li><li> 샙터 세포 카운터는 다음 세포 농도를 결정하는 데 사용되었다. 그림 1은 이론적으로 세포 농도와 함께 오 대표 세포 라인에 대한 측정되었다 5 dilutions를 보여줍니다. </li><li> 로 R2의 값을로 표시 선형의 높은 정도, 그 샙터 카운트가 넓은 선형 작동 범위에 걸쳐, 테스트 세포 라인에 대한 안정적인 방법입니다 보여줍니다. </li><li> 샙터 계수의 전반적인 상대 정확도는 샙터 계산에서 얻은 데이터가 쿨터 계산에서 얻은 계산과 비교하는 그림 2에서 증명됩니다. </li><li> 샙터 셀 카운트의 로그 쿨터 카운트의 로그 역모를 꾸몄다하고, 테스트를 모든 세포 라인에 걸쳐, 샙터 카운트가 쿨터 카운트와 일치하는, 표시합니다. </li><li> 19 셀 라인 카운트는 Z2 쿨터 카운터, 샙터 셀 카운터와 같은 바이올렛 - 셀 또는 셀 시스템과 같은 자동화된 비전 기반의 카운터, 그리고 hemocytemeter를 사용하여 수행되었습니다. </li><li> 카운트는 정지와 동일한 dilutions를 시작하는 동일한 세포를 사용하여 제조 업체의 지시에 따라 수행되었다. 그림 3에 표시된 결과는 긴밀하게 높은 선형성과 함께 이론적으로 세포 농도와 일치 샙터는 계산에 의해 측정되는 세포 농도를 보여줍니다. </li><li> 퍼센트 편차 계수, 또는 CV는 각각 집계 시스템에 대한 각 세포주의 각 희석 계산되었다. 그림 4는 세포 농도 및 계산 방법에 관한 19 셀 라인을 통해 평균 %의 CVS를 보여줍니다. </li><li> 작동 범위 내에서 샙터 전지 카운터 작은 표준 편차와 변동 작은 평균 계수를 표시 비전을 기반으로 hemocytometer의 계산보다 더 정확합니다. </li><li> 샙터 계산 열 배 hemocytometry보다 너무 다른 자동 카운터보다 빠릅니다. 그림 5, SF9, MCF7, 그리고 HEK293 세포의이 동영상에서 설명한 방법을 사용하여 계산되었다. </li><li> 세포 농도를 측정하는 데 필요한 평균 시간이 기록되었다. hemocytometry은 샘플 당 한 번만 수행이라고합니다. </li><li> 샙터는 셀 크기에 따라 측정합니다. 따라서 세포 형태의 변화는 히스토그램에서 캡처 수 있습니다. 건강하지 못한 세포는 lyse, 그리고 왼쪽에있는 히스토그램의 변화 (그림 6, 왼쪽 패널)을 일으킬 동안 건강한 문화는 좋은 종 모양의 가우스 분포를 표시합니다. </li><li> 마찬가지로 50 %의 죽은 세포 인구가 50% 라이브 세포와 혼합되었을 때, 히스토그램 교대도 (그림 6, 오른쪽 패널) 관찰되었다. </li></ol><img src="/files/ftp_upload/2204/2204fig1.jpg" alt="그림 1" /><img src="/files/ftp_upload/2204/2204fig2.jpg" alt="그림 2" /><img src="/files/ftp_upload/2204/2204fig3.jpg" alt="그림 3" /><img src="/files/ftp_upload/2204/2204fig4.jpg" alt="그림 4" /><img src="/files/ftp_upload/2204/2204fig5.jpg" alt="그림 5" /><img src="/files/ftp_upload/2204/2204fig6.jpg" alt="그림 6" /><img src="/files/ftp_upload/2204/2204fig7.jpg" alt="그림 7" />

<ol>
	<li>Barghshoon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Counting Survey. , (2009).</li><li>Tucker, K. G., Chalder, S., al-Rubeai, M., Thomas, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+hybridoma+cell+number,+viability,+and+morphology+using+fully+automated+image+analysis.">Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis.</a> Enzyme Microb Technol. 16 (1), 29-35 (1994).</li><li>Houwen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fifty years of hematology innovation: the Coulter principle. , (2003).</li></ol>

캐슬린 Ongena, Chandreyee 다스, 재닛 L. 스미스, 소니아 길, 그리고 그레이스 존스톤이 문서에서 사용되는 악기를 생산 Millipore에 의해 고용됩니다.

다른 계산 방법의 결과에 샙터 셀 카운터의 성능을 비교, 우리는이 새로운 휴대용, 자동 셀 카운터가 다양한 작동 범위에서 정확, 신속하고, 안정적인 셀 카운트를 제공 결론. 샙터 계수의 기능은 센서 팁 및 쿨터 원칙에 따라 정교한 계산 장비에 내장된 정밀 설계 기술의 결과입니다. 성능 품질은 속도를 증가시키고 세포 기반 assays의 재현성을 향상시키는 방법으로 계산 샙터을 제안합니다. 핸드헬드 형식은 세포 배양 후드에서 빠른 지겨움 무료로 집계 권리를 허용합니다.

<table border="1"><tbody><tr><td> 재료 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 논평 </td></tr><tr><td> 40 μm의의 샙터 센서와 샙터 2.0 휴대용 자동 전지 카운터 </td><td> EMD Millipore </td><td> PHCC20040 </td><td></td></tr><tr><td> 60 μm의 샙터 센서와 샙터 2.0 휴대용 자동 전지 카운터 </td><td> EMD Millipore </td><td> PHCC20060 </td><td></td></tr><tr><td> 샙터 센서, 60 μm의 </td><td> EMD Millipore </td><td> PHCC60050 </td><td> 센서 50 팩 </td></tr><tr><td> 샙터 센서, 40 μm의 </td><td> EMD Millipore </td><td> PHCC40050 </td><td> 센서 50 팩 </td></tr><tr><td> 범용 전원 어댑터 </td><td> EMD Millipore </td><td> PHCCPOWER </td><td> </td></tr><tr><td> 샙터 O - 링 키트 </td><td> EMD Millipore </td><td> PHCCOCLIP </td><td> 이 O - 링과 하나의 필터 커버 </td></tr></tbody></table>

determining cell number during cell culture using the scepter cell counter

세포를 계산하는 것은 자주 체외 세포 문화에 대한 필요하지만 지루한 단계입니다. 일관된 세포 농도는 실험 재현성과 정확성을 보장합니다. 셀 카운트는 후속 실험, transfection 또는 감염 세포를 심는 불멸화 또는 변환을 평가, 세포 건강과 확산 속도를 모니터링하고, 셀 기반의 assays를위한 준비 중요합니다. 셀 카운트 특히 세포 반응의 양적 측정, 정확한 일관성 및 빠른 것이 중요합니다. 셀 카운팅의 속도와 정확성이 필요에도 불구하고, 400 연구자의 ​​71 %는 hemocytometer를 사용하여 세포를 카운트 1 조사. hemocytometry 저렴한지만, 그것은 힘드는과 부정 확한 계산 결과를 사용자의 바이어스 및 남용에 적용됩니다. Hemocytometers는 셀 suspensions가 지정된 볼륨에로드하고 현미경으로 집계하고있는 특수 광학 유리로 만들어진 있습니다. hemocytometry에서 오류의 소스는 다음과 같습니다 : 예제에서 고르지 세포 배포 예제에서 너무 많은 혹은 너무 적은 세포, 특정 세포가 정의된 계산 영역에 속하는 것인 지에 대한 주관적인 의사 결정, hemocytometer의 오염, 사용자에게 사용자의 변화를 그리고 hemocytometer의 변형 속도 2 작성. 수동 계산과 관련된 지겨움을 완화하기 위해, 연구자의 ​​29 %가 장치를 계산 자동 세포를 사용하여 세포를 포함, 이들은 비전 기반 카운터, 쿨터 원리를 사용하여 세포를 감지 시스템, 또는 유동세포계측법 1 포함됩니다. 대부분의 연구자 들어, 자동화된 시스템을 사용하는 주요 장벽이 큰 benchtop 악기 하나와 관련된 가격입니다. 샙터 전지 카운터는 상대적으로 낮은 비용으로 자동화 및 쿨터 계산의 정확도를 제공하는 자동화된 휴대용 장치입니다. 시스템은 쿨터 감지를위한 아날로그 및 디지털 하드웨어의 조합을 사용하여 소형 형식으로 임피던스 기반의 입자 검출 3 원칙, 신호 처리, 데이터 스토리지 및 그래픽 디스플레이를 고용합니다. 일회용 팁은 세포의 크기와 서브 마이크론과 하위 picoliter 해상도에서 셀 볼륨에 의해 차별을 가능하게 microfabricated, 셀 감지 영역에 설계되었습니다. 정밀 액체 처리 채널과 전자와 향상된, 샙터 전지 카운터 그래픽 히스토그램으로 표시 셀 인구 통계를보고합니다.

Watch this Scientific Journal Video about Determining Cell Number During Cell Culture using the Scepter Cell Counter at JoVE.com

Determining Cell Number During Cell Culture using the Scepter Cell Counter

샙터 셀 카운터, 세포를 카운트 세포 직경과 볼륨을 모니터링 한 문화에서 다음 세포 인구의 건강과 품질을 검사하는 데 사용하는 데 사용할 수있는 휴대용 자동 장치입니다.

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 ...

determining-cell-number-during-cell-culture-using-scepter-cell

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JoVE Journal

Biology

37.8K 조회수.  Millipore Inc. 샙터 셀 카운터, 세포를 카운트 세포 직경과 볼륨을 모니터링 한 문화에서 다음 세포 인구의 건강과 품질을 검사하는 데 사용하는 데 사용할 수있는 휴대용 자동 장치입니다.

비디오: Determining Cell Number During Cell Culture using the Scepter Cell Counter

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

냉동 세포에서 시작 문화 : 인간 ES 세포

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

연구

생물학

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

유전자 변형으로 인코딩된 칼슘 센서를 사용하여 Apoptosis 동안 Mitochondrial 칼슘 레벨에서 동적 변경 사항 모니터링

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral - 중재 최저 동안 예 VIVO</em인간 조혈 줄기 세포의> 적혈구 조혈

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

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 ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Zebrafish 개발하는 동안 Photoconvertible 단백질을 사용하여 셀 추적

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Blastomere Explants는 배아 개발하는 동안 세포 운명의 헌신에 대해 테스트 할

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

유방암 세포주에서 세포 증식을 결정하기위한 세 가지 방법 비교

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

라이브 세포 형광 현미경 검사 법 미생물 세포 성장 하는 동안 필수 과정을 관찰 하

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

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.