위에서 설명한 기존 연구는 NSF 교부금 0,344,630에 의해 지원되었다.

<ol>
	<li>Lochhead, J. H., Brown, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+Invertebrate+Types.">Selected Invertebrate Types.</a> , 360-381 (1950).</li><li>Shuster, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circulatory+system+and+blood+of+the+horseshoe+crab+Limulus+polyphemus+L.:+a+review.">The circulatory system and blood of the horseshoe crab Limulus polyphemus L.: a review.</a> US Dept Energy, Fed Regul. Comm. , (1978).</li><li>Conrad, M. L., Pardy, R. L., Wainwright, N., Child, A., Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+the+blood+clotting+system+of+the+American+horseshoe+crab,+Limulus+polyphemus,+to+a+novel+form+of+lipopolysaccharide+from+a+green+alga.">Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga.</a> Comp Biochem. Physiol A Mol. Integr. Physiol. , 144-423 (2006).</li><li>Nakamura, T., Morita, T., Iwanaga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+proclotting+enzyme+in+limulus+(Tachypleus+tridentatus)+hemocytes:+its+purification+and+properties.">Intracellular proclotting enzyme in limulus (Tachypleus tridentatus) hemocytes: its purification and properties.</a> J. Biochem. (Tokyo. 97, 1561-1574 (1985).</li><li>Levin, J., Ornberg, R. L., Cohen, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+Cells+of+Marine+Invertebrates:+Experimental+Systems+in+Cell+Biology+and+Comparative+Physiology.">Blood Cells of Marine Invertebrates: Experimental Systems in Cell Biology and Comparative Physiology.</a> , 259-260 (1985).</li><li>Liang, S. M., Liu, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+Limulus+coagulation+system:+inhibition+of+activation+of+the+proclotting+enzyme,+by+dimethyl+sulfoxide.">Studies on the Limulus coagulation system: inhibition of activation of the proclotting enzyme, by dimethyl sulfoxide.</a> Biochem. Biophys. Res. Commun. 105, 553-559 (1982).</li><li>Soderhall, K., Smith, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+the+haemocyte+populations+of+Carcinus+maenas+and+other+marine+decapods,+and+prophenoloxidase+distribution.">Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution.</a> Dev. Comp Immunol. 7, 229-239 (1983).</li><li>Solon, E., Gupta, A. P., Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+during+exocytosis+in+Limulus+polyphemus+granulocytes.">Signal transduction during exocytosis in Limulus polyphemus granulocytes.</a> Dev. Comp. Immunol. 20, 307-321 (1996).</li><li>Armstrong, P. B., Rickles, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin-induced+degranulation+of+the+Limulus+amebocyte.">Endotoxin-induced degranulation of the Limulus amebocyte.</a> Exp. Cell Res. 140, 15-24 (1982).</li><li>Iwanaga, S., Kawabata, S., Shuster, C. N., Barlow, R. B., Berkman, L., Brockmann, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+phylogeny+of+defense+molecules+associated+with+innate+immunity+in+horseshoe+crab.">Evolution and phylogeny of defense molecules associated with innate immunity in horseshoe crab.</a> Front Biosci. 3, (1998).</li><li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+American+Horseshoe+Crab.">The American Horseshoe Crab.</a> , 288-309 (2003).</li><li>Iwanaga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+limulus+clotting+reaction.">The limulus clotting reaction.</a> Curr. Opin. Immunol. 5, 74-82 (1993).</li><li>Levin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Limulus+amebocyte+lysate+test:+perspectives+and+problems.">The Limulus amebocyte lysate test: perspectives and problems.</a> Prog. Clin. Biol. Res. 231, 1-23 (1987).</li><li>Koshiba, T., Hashii, T., Kawabata, 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+structural+perspective+on+the+interaction+between+lipopolysaccharide+and+factor+C,+a+receptor+involved+in+recognition+of+Gram-negative+bacteria.">A structural perspective on the interaction between lipopolysaccharide and factor C, a receptor involved in recognition of Gram-negative bacteria.</a> J. Biol. Chem. 282, 3962-3967 (2007).</li><li>James, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+protease+inhibitor+phenylmethylsulfonyl+fluoride+in+buffers.">Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers.</a> Anal. Biochem. 86, 574-579 (1978).</li><li>Murer, E. H., Levin, J., Holme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+studies+of+the+granules+of+the+amebocytes+of+Limulus+polyphemus,+the+horseshoe+crab.">Isolation and studies of the granules of the amebocytes of Limulus polyphemus, the horseshoe crab.</a> J. Cell Physiol. 86, 533-542 (1975).</li><li>Decker, H., Ryan, M., Jaenicke, E., Terwilliger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS+induced+phenoloxidase+activity+of+hemocyanins+from+Limulus+polyphemus,+Eurypelma+californicum+and+cancer.">SDS induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum and cancer.</a> , 276-17796 (2001).</li><li>Armstrong, P. 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+cytolytic+function+for+a+sialic+acid-binding+lectin+that+is+a+member+of+the+pentraxin+family+of+proteins.">A cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins.</a> J. Biol. Chem. 271, 14717-14721 (1996).</li><li>Armstrong, P. B., Melchior, R., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humoral+immunity+in+long-lived+arthropods.">Humoral immunity in long-lived arthropods.</a> J. Insect Physiol. 42, 53-64 (1996).</li><li>Armstrong, P. B., Armstrong, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+decorated+clot:+Binding+of+agents+of+the+innate+immune+system+to+the+fibrils+of+the+limulus+blood+clot.">The decorated clot: Binding of agents of the innate immune system to the fibrils of the limulus blood clot.</a> Biol. Bull. 205, 201-203 (2003).</li><li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+the+motile+blood+cells+of+the+horseshoe+crab,+Limulus.+Studies+on+contact+paralysis+of+pseudopodial+activity+and+cellular+overlapping+in+vitro.">Interaction of the motile blood cells of the horseshoe crab, Limulus. Studies on contact paralysis of pseudopodial activity and cellular overlapping in vitro.</a> Exp. Cell Res. 107, 127-138 (1977).</li><li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+of+the+Limulus+blood+cell.">Motility of the Limulus blood cell.</a> J. Cell Sci. 37, 169-180 (1979).</li><li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+and+spreading+of+Limulus+blood+cells+on+artificial+surfaces.">Adhesion and spreading of Limulus blood cells on artificial surfaces.</a> J. Cell Sci. 44, 243-262 (1980).</li><li>Armstrong, P. B., Cohen, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+Cells+of+Marine+Invertebrates.">Blood Cells of Marine Invertebrates.</a> Lab. Animal. , 253-258 (1985).</li><li>Smith, S. A., Berkson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+culture+and+maintenance+of+the+horseshoe+crab+(Limulus+polyphemus).">Laboratory culture and maintenance of the horseshoe crab (Limulus polyphemus).</a> Lab. Animal. 34, 27-34 (1980).</li><li>Leibovitz, L., Lewbart, G. A., Shuster, C. N., Barlow, R. B., Brockmann, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+American+Horseshoe+Crab.">The American Horseshoe Crab.</a> , 245-275 (2003).</li><li>Rudkin, D. M., Young, G. A., Nowlan, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oldest+horseshoe+crab:+a+new+xiphosurid+from+Late+Ordovician+Konservat-Lagerstatten+deposits.">The oldest horseshoe crab: a new xiphosurid from Late Ordovician Konservat-Lagerstatten deposits.</a> Palaeontology. 51, 1-9 (2008).</li><li>Shuster, C. N., Sekiguchi, K., Shuster, C. N., Barlow, R. B., Brockmann, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The American Horseshoe Crab. , 103-132 (2003).</li><li>Walls, E. A., Berkam, J., Smith, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+horseshoe+crab,+Limulus+polyphemus,+200+million+years+of+existence,+100+years+of+study.">The horseshoe crab, Limulus polyphemus, 200 million years of existence, 100 years of study.</a> Rev. Fisheries Sci. 10, 39-73 .</li><li>Patten, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Evolution+of+the+Vertebrates+and+Their+Kin.">The Evolution of the Vertebrates and Their Kin.</a> P. Blakiston's Son and Co. , 195-209 (1912).</li></ol>

말굽 게 4 종의, 동쪽의 북아메리카 해안 세 종 일본에서 벵골 만 해당 범위에서 Limulus polyphemus의가 있습니다. 해부학적인 몸의 구조와 유사한 형태가 27 4억4천5백만년 전에서 화석으로 발견 되었기 때문에 동물은 &quot;살아있는 화석&quot;으로 식별됩니다. 성숙에 도달 13 년 최소한 20 년은 28 생활 - 말굽 게 10을 필요로하는, 긴 동물을 나타냅니다. 그것은 장어와 콘치 어업 29 미끼로 ?...

blood collection from the american horseshoe crab, limulus polyphemus

말굽 게 어떤 긴 무척추 동물의 가장 특징 면역 체계가 있습니다. 말굽 게는에서 면역의 연구는 혈액의 대량 수집 용이성으로하고 혈액의 단순에서 촉진되었다. 말굽 게는 일반적인 순환, 세분화 amebocyte에서만 하나의 셀 유형을 보여줍니다. 플라즈마는 바닷물의 염분 성분을 가지고 있으며, 단지 세 풍부한 단백질, hemocyanin, 호흡기 단백질, C - 반응 박테리아 세포를 포함한 외부 세포의 cytolytic 파괴 기능 단백질, 및 α2 - macroglobulin은 프로 테아제를 억제하는 병원균을 침해니다. 혈액은 lipopolysaccharide (일명, 내독소, LPS), 그람 음성 세균의 제품으로 오염을 최소화 조건 하에서 직접 심장 주사에 의해 수집됩니다. 혈액 400 ML - 대형 동물은 200를 얻을 수 있습니다. 플라즈마의 연구, 혈액 세포는 즉시 원심 분리에 의해 혈장에서 제거되며 플라즈마 다음의 구성 단백질로 fractionated 수 있습니다. 혈액 세포가 편리하게 다음 그 coverglasses를 장착, 문화 접시 표면에 대한 자세한 중 하나 LPS 무료 coverglasses를 삽입하여 직접 현미경 검사를 허용 조건 LPS 무료 isotonic 호수 (0.5 M NaCl)에 혈액 작은 볼륨을 수집하여 미세 공부 아르 세포 부착 다음과 같은 간단한 관측 실 인치 직접 관찰을위한 두 번째 준비 3를 수집하는 것입니다 - LPS없는 배아 요리의 혈액 5 ML하고 다음 실로 총체적 amebocytes의 조각을 explanting하는 슬라이드와 coverglass 사이에 샌드위치가 조직. 이 준비에서 운동성이있는 amebocytes 그들이 쉽게 볼 수 있습니다 coverglass 표면에,에 마이 그 레이션. 혈액 응고 시스템 amebocytes의 집합과 혈액 세포의 분비 과립의에서 발표되는 단백질, coagulin의 세포외 응고의 형성을 포함한다. 세탁 혈액 세포의 생화 학적 분석 2퍼센트 트윈 - 20, 0.5 M LPS 무료 NaCl, 세포의 원심 분리에 의해 다음과 0.5로 세척 M의 0.1 볼륨으로 혈액을 수집하여 수행할 수 수 집합과 degranulation가 발생하지 않습니다을 필요로 NaCl.

Watch this Scientific Journal Video about Blood Collection from the American Horseshoe Crab, Limulus Polyphemus at JoVE.com

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

미국 말굽 게, Limulus polyphemus은 틀림없이 모든 무척추 동물의 혈액의 다량을 위해 가장 편리한 소스입니다. 혈액의 일반적인 순환, 세분화 amebocyte, 그리고 플라즈마, hemocyanin, C - 반응 단백질 및 α2 - macroglobulin에서 3 풍부한 단백질에 하나의 셀 유형, 구성의 간단하다. 혈액은 심장에서 수집되고 혈액 세포 및 플라즈마 원심 분리로 구분됩니다.

미국 호르세쇼 게, Limulus Polyphemus의 혈액 수집

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

blood-collection-from-the-american-horseshoe-crab-limulus-polyphemus

Research

JoVE Journal

Biology

29.9K 조회수.  University of California, Davis.  미국 말굽 게, Limulus polyphemus은 틀림없이 모든 무척추 동물의 혈액의 다량을 위해 가장 편리한 소스입니다. 혈액의 일반적인 순환, 세분화 amebocyte, 그리고 플라즈마, hemocyanin, C - 반응 단백질 및 α2 - macroglobulin에서 3 풍부한 단백질에 하나의 셀 유형, 구성의 간단하다. 혈액은 심장에서 수집되고 혈액 세포 및 플라즈마 원심 분리로 구분됩니다. 

비디오: Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

StemCell 기술에서 RosetteSep 키트와 함께 인간의 피를에서 NK 세포의 농축

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

연구

생물학

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

The American horseshoe crab, Limulus Polyphemus is one of the oldest creatures on earth, and the animal continues to play an indispensable role in biomedical research. Not only does their blood contain special cells that scientists use to detect bacteriotoxins in our medicines, but their eyes also contain a neural network that has provided much insight about physiological processes operating in our visual system, such as light adaptation and lateral inhibition. The horseshoe crab remains an attractive model for vision research because the animal is large and hardy for an invertebrate, its retinal neurons are big and easily accessible, its visual system is compact and extensively studied, and its visual behavior is well defined. Moreover, the structure and function of the eyes are modulated on a daily basis by a circadian clock in the animal s brain. In short, the visual system of horseshoe crabs is simple enough to be understood yet complex enough to be interesting.
In this video we present three electrophysiological paradigms for investigating the neural basis of vision that can be performed in vivo with Limulus. They are electroretinogram recording, optic nerve recording, and intraretinal recording. Electroretinogram (ERG) recordings measure with a surface electrode the summed electrical response of all cells in the eye to a flash of light. They can be used to monitor the overall sensitivity of the eye for prolong periods of time. Optic nerve recordings measure the spiking activity of single nerve fibers with an extracellular microsuction electrode. They can be used to study visual messages conveyed from the eye to the brain as well as circadian-clock messages fed back from the brain to the eye. Intraretinal recordings measure with an intracellular microelectrode the voltage fluctuations induced by light in individual cells of the eye. They can be used to elucidate cellular mechanisms of retinal processing.

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

In this video we perform electroretinogram recording, optic nerve recording, and intraretinal recording with the American horseshoe crab, Limulus Polyphemus. These electrophysiological paradigms can be used for investigating the neural basis of vision in a research or teaching lab.

호르세쇼 크랩을 사용하여, Limulus Polyphemus 비전 연구

The American horseshoe crab, Limulus Polyphemus is one of the oldest creatures on earth, and the animal continues to play an indispensable role in ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

신생아 마우스의 폐 내피 세포의 분리 및 문화

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

성인 Zebrafish의 생물 화학 분석을위한 혈액 수집

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

플라즈마 단백질 보존을위한 혈액 수집 장비로부터 게놈 DNA를 추출하는 실제적이고 새로운 방법

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

당연히 환경에서 Magnetotactic 박테리아를 발생의 수집, 절연 및 강화

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

에피 솜과 HDAC 억제제를 사용하여 혈액에서 효율적인 유도 만능 줄기 세포 생성

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

쥐의 측면 꼬리 정맥에서 혈액을 샘플링

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

성인 Zebrafish의에서 혈액 검사에 대한 반복 채혈

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

마우스에서 대뇌 골후혈구를 분리하는 고출력 방법

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...