Teitell 연구실에서 인간 배아 줄기 세포 연구는 재생 의료에 대한 캘리포니아 연구소 (CIRM) 씨앗 부여 RS1 - 00313에 의해 지원됩니다. 우리는 UCLA, 특히 박사 Amander 클라크 박사 제롬 잭, 우리의 연구들의 지원 UCLA 브로드 연구소 줄기 세포 핵심 시설의 멤버에 재생 의료 및 줄기 세포 연구의 브로드 센터의 회원을 감사드립니다.

Matrigel에서 Matrigel을 분리 hESCs 보통 Matrigel에 hESCs의 합류 6 잘 판은 우물이 4-5일 분리 후 다시 합류되기와 다른 Matrigel 판에 1시 5분에서 1시 3분까지 분할 수 있습니다. <ol><li> 분할하기 전에 위에서 설명한 CM과 Matrigel 플레이트는 준비가되어 있습니다. </li><li> 분할 당일, × 1 분리를위한 PBS, pH7.4를 각각 잘 씻어 각각 잘하는 1mg/ml Dispase (DMEM/F12 매체와 희석 5mg/ml 재고 솔루션)의 1ml을 추가하고, 37 품어 ° C 3 분. </li><li> 1 각 잘 세 번 씻어 × PBS, pH7.4은, bFGF없이 ES 미디어 1ml를 추가 우물의 바닥에서 식민지를 긁어 세포 스크레이퍼를 사용하여 50ml 팔콘 튜브의 정지 hESCs를 수집하고, 원심 분리에 의해 펠릿 200g에 상온에서 5 분. </li><li> 다시 타석으로 Matrigel 플레이트에 hESCs 먼저 DMEM/F12과 Matrigel 접시를 씻어 잘 당 10ng/ml bFGF와 보충 CM의 2.5ml를 추가합니다. 10ng/ml bFGF, 피펫 세포 현탁액 위, 아래로 세 번 함께 보충 CM의 적절한 볼륨에 hESC 펠렛을 Resuspend. (참고 : 분리를위한 hESCs가 Matrigel 접시에서 온 때, 대단히 짧은 휴식을 매우 쉽게 않으므로 pipetting 3 번을 충분하고 더 식민지를 오버 방해와 단일 세포를 만들 수 있습니다 pipetting.) 나누어지는은 당 hESC의 대단히 짧은 시간 서스펜션 0.5 ML 잘 6 잘 Matrigel 판의 최종 볼륨으로 당 3ml 잘 달성하기 위해. 37 ° C 배양기에서 접시를 놓습니다. </li></ol> immunofluorescence 현미경에 의해 pluripotency 마커 감지 Immunofluorescence 현미경은 culturing 동안 hESC pluripotency 마커 월 4 SSEA - 4의 표현을 확인하는 데 사용됩니다. <ol><li> 잘 하나에서 미디어를 기음과 1 × PBS 세척, 산도 7.4. 10 분에 대한 수정을 잘하기 위해 3.7 %의 포름 알데히드 2ml를 추가합니다. </li><li> 두 분 -20에서 잘과 장소 ° C에 2ml 메탄올을 추가 1xPBS, pH를 7.4로 씻어. </li><li> 5 분 알부민 0.2 % 소 serume (BSA)의 1ml와 1xPBS, 산도 7.4, 그리고 블록 린스. </li><li> 월 4 anti-h/mSSEA-4 항체 (0.2 % BSA에 1:200 희석)의 1ml을 추가하고, 1 시간 또는 4 ° C에서 하룻밤 실온에서 알을 품다. (참고 : SSEA - 4 염색법을위한 6 단계로 바로 진행합니다) </li><li> , 1xPBS, pH를 7.4로 세 번 씻어 FITC - 복합 토끼 IgG (0.2 % BSA에 1:500 희석), 그리고 1 시간에 대한 실온에서 부화를 추가합니다. </li><li> 1xPBS, 산도 7.4 3 번 씻어하고 잘의 중심에 (핵 시각화가 원하는 경우 DAPI를 포함할 수 있습니다) 장착 솔루션의 한 방울을 넣어. 매니큐어와 인감, 세포의 상단에 coverslip을 넣어, 그리고 immunofluorescence 현미경에 의해 평가합니다. </li></ol> 인간 배아 줄기 세포 영수증 ES 미디어 (배아의 줄기 세포 미디어) : DMEM/F12 - 400ml 마네 세럼 Replacer - 100ml 비 - 필수 아미노산 - 5ml 200mM GlutaMax / BME 솔루션 - 5ml 페니실린 / 스트렙토 마이신 - 5ml 4 잘 섞어 500ml 병의 최종 hESC 문화 미디어를 저장 ° C. 2 주간 최대 좋아. MEF 문화 미디어 : DMEM - 1000ml FBS - 100ml 비 - 필수 아미노산 - 10ml 글루타민 - 10ml Penicllin / 스트렙토 마이신 - 10ml 잘 믹스 4에서 최종 MEF 문화 미디어를 저장 ° C. 200mM Glutamax / BME 솔루션 : Glutamax - 100ml β - 메르 캅 토 에탄올 (14.3M에 증권 솔루션) - 140 μ1 이 잘 그리고 솔루션 나누어지는의 10ml의 일부를 섞는다. -20 ° C.에 aliquots 유지 bFGF 솔루션 (재고의 최종 농도 : 10 마이크로 그램 / ML) : bFGF (FGF2) - 50 μg 1xPBS에 0.​​1 % BSA, 산도 7.4 - 5ml 10 μg / ML 주식을하기 위해 5ml 0.1 % BSA에 bFGF 50 마이크로 그램을 디졸브. 500 μl 부분으로 나누어지는. -80에서 보관 ° C. Collagenase IV 솔루션 (1mg/ml) : Collagenase IV - 50mg DMEM/F12 미디어 - 50ml 50ml 팰컨 튜브에 collagenase IV 가루를 추가합니다. 후드에서 무균 DMEM/F12 미디어 50ml에 섞는다. &lt;1 분 소용돌이 솔루션입니다. 가루가 해산되어 있는지 확인합니다. 후드에서 새 살균 50ml 팔콘 튜브에 필터 0.22 μm의 통과. 이 솔루션은 4 저장된 이주의 최대 ° C.에 좋습니다 Dispase 솔루션 (1mg/ml) : Dispase 5mg/ml - 2ml DMEM/F12 미디어 - 8ml -20 ° C.에서 나누어지는 상업 5mg/ml의 Dispase 솔루션과 저장 DMEM/F12를 사용 1mg/ml로 dispase을 희석. 이 솔루션은 4에서 1 주일 동안 보관 가능 ° C.

<ol>
	<li>Thomson, J. a. m. e. s. A., Itskovitz-Eldor, J. o. s. e. p. h., Shapiro, S. a. n. d. e. r. S., Waknitz, M. i. c. h. e. l. l. e. A., Swiergiel, J. e. n. n. i. f. e. r. J., Marshall, V. i. v. i. e. n. n. e. S., Jones, J. e. f. f. r. e. y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts+.">Embryonic Stem Cell Lines Derived from Human Blastocysts .</a> Science. 282, 1145-1147 (1998).</li><li>Xu, C. h. u. n. h. u. i., Inokuma, M. a. r. g. a. r. e. t. S., Denham, J. e. r. r. o. d., Golds, K. a. t. h. a. l. e. e. n., Kundu, P. r. a. t. i. m. a., Gold, J. o. s. e. p. h. D., Carpenter, M. e. l. i. s. s. a. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells+Nature+Biotechnology+19."> Feeder-free growth of undifferentiated human embryonic stem cells Nature Biotechnology 19.</a> Nature Biotechnology. 19, 971-974 (2001).</li></ol>

3 동영상이 시리즈는 (MEF) 피더 세포 (비디오 1) 어떻게 통로를 피더 셀 - 무료 접시 (비디오 2) Matrigel을하고, 방법을 유지하기 위해 마우스 배아 fibroblast에서 인간 배아 줄기 세포 (hESCs)를 성장하는 방법을 보여줍니다 Matrigel 피더 셀 무료 조건 (동영상 3) passaging하여 hESCs. 많은 이전의 연구는 실용적, undifferentiated hESCs의 유지 보수가 inactivated MEF 피더 세포에 대한 문화를 필요로했다. 그러나 대부분의 실험, 피더 세포 오염의 무료 hESCs의 순수한 인구가 필요합니다. 이러한 목표를 달성하기 위해, 우리는 어떻게 통과 hESCs에 MEF 피더 플레이트에서 피더 셀 - 무료 Matrigel 플레이트 및 유지하고 피더에서 연속 통과 hESC 식민지 세포가없는 조건을 증명하고있다. 이 프로토콜에 따라, MEF 피더 셀 오염의 작은 금액은 아직 Matrigel의 접시에 통로 하나가 존재할 수 있지만,이 오염 물질은 일반적으로 시각화하기 쉽습니다. 이상 Matrigel 통로 둘 때, 아무 MEF 오염은 일반적으로 실험에 대한 hESCs의 순수한 인구를 떠나 발견되지 않습니다. 그러한 월 4 SSEA - 4로 hESC pluripotency 마커에 대한 Immunofluorescence 염색법과 현미경이나 흐름 cytometry는 문화 중에 undifferentiated 상태에서 hESCs의 유지를 확인하기 위해 필요합니다.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Knockout Serum Replacer (KSR)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td> Non-essential Amino Acids</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMax </td>
<td>Reagent</td>
<td>Gibco</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11995-065</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>Reagent</td>
<td>Clontech</td>
<td>631107</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25030-081</td>
<td>&nbsp;</td>
<tr>
<td>BME</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP176-100</td>
<td>&nbsp;</td>
<tr>
<td>bFGF</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>233-FB-025</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV</td>
<td>Reagent</td>
<td>Gibco</td>
<td>17104-019</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>Reagent</td>
<td>Stem Cell Technologies</td>
<td>17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin / Streptomycin </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin</td>
<td>Reagent</td>
<td>Chemicon</td>
<td>ES-006-B</td>
<td>&nbsp;</td>
<tr>
<td>Matrigel</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>354277</td>
<td>&nbsp;</td>
<tr>
<td>Oct-4 antibody</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>SC-9081</td>
<td>&nbsp;</td>
<tr>
<td>anti-h/mSSEA-4 Phycoerythrin Conjugated Mouse IgG3</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>FAB1435P</td>
<td>&nbsp;</td>
<tr>
<td>FITCI-conjugated antirabbit IgG</td>
<td>Reagent</td>
<td>Jackson ImmunoResearch Laboratories. Inc.</td>
<td>715-095-152</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
					</div>

from mefs to matrigel 3: passaging hescs from matrigel onto matrigel

이 동영상은 피더 세포가없는 조건에서 어떻게 피더에서 연속 통과 hESCs 세포가없는 조건에 인간 배아 줄기 세포 (hESCs)의 성장을 유지하는 방법을 보여줍니다. hESC의 확인은 pluripotency immunofluorescence 현미경으로 피더 셀없는 조건을 재배도 증명됩니다.

Watch this Scientific Journal Video about From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel at JoVE.com

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

이 동영상은 피더 세포가없는 조건에서 어떻게 피더에서 연속 통과 hESCs 세포가없는 조건에 인간 배아 줄기 세포 (hESCs)의 성장을 유지하는 방법을 보여줍니다. hESC의 확인은 pluripotency immunofluorescence 현미경으로 피더 셀없는 조건을 재배도 증명됩니다. 3 부 3.

MEFs에서 Matrigel 3 : Matrigel에 Matrigel에서 Passaging hESCs

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

from-mefs-to-matrigel-3-passaging-hescs-from-matrigel-onto-matrigel

Nanyang Technological University

Research

JoVE Journal

Biology

13.7K 조회수.  University of California, Los Angeles. 이 동영상은 피더 세포가없는 조건에서 어떻게 피더에서 연속 통과 hESCs 세포가없는 조건에 인간 배아 줄기 세포 (hESCs)의 성장을 유지하는 방법을 보여줍니다. hESC의 확인은 pluripotency immunofluorescence 현미경으로 피더 셀없는 조건을 재배도 증명됩니다. 3 부 3.

비디오: From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

트립신과 Passaging 색채 인간의 배아 줄기 세포 - 라인

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

연구

생물학

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

인간 신경 줄기 세포를 Passaging

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

MEFs부터를 Matrigel하기 : MEFs의 면전에서 Passaging hESCs를

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

삭스 - 2의 강제 표현을 통해 MEFS에서 IPS 세포를 생성, 10 - 4, C - Myc, 그리고 Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

Matrigel에 MEFs에서 분리 hESCs : MEFs에서 Matrigel 2

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

Kinesin 전원 분자 셔틀로화물 로딩

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

웜 실험실 소개 : Culturing 웜에서 돌연변이 유발하는

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

종양 세포의 Vasculogenic 활동을 평가 Matrigel 기반 튜브 형성 분석

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

의 효율적인 생산 및 정제 재조합 쥐과 Kindlin-3 생물 물리학 연구 곤충 세포에서

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

성적인 프로그램을 다시 만들에 의해 인간 배아 줄기 세포에서 Myospheres의 생성

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...