자금 출처 : SVN에 코네티컷 연구 재단과 분자 및 세포 생물학의학과 대학교, 시그마 사이 보조금 수준의 연구와 안토니오 H. 및 마조리 R. AJC에 로마노 대학원 교육 원정대

<ol><li> 0.22 μm의 필터 살균 인공 해수 500 MLS (; 염도 35 PPT F​​SW)를 준비합니다. 0.22 μm의 미크론 필터를 통해 필터 인공 또는 자연 해수는 입자와 세균을 제거합니다. </li><li> FSW의 에탄올의 2 % 용액에 배치하여 한 성인 하와이 밥테일 오징어 (Euprymna scolopes)을 마취. 약 10 분 동안 마취에서 동물을 놓으십시오. 오징어 수영을 중지하고 적극적으로 터치에 응답하지 않습니다. 계속 호흡, 맨틀의 움직임에 의해 표시하고, chromatophore 활동은 계속 관찰하여야한다. </li><li> 표준 왁스 절개 트레이에 직면 복부 측면과 오징어를 넣으십시오. FSW 2 %의 에탄올을 포함하는으로 동물 잠수함. </li><li> 하나의 표준 200 μl 피펫 팁을 사용하여 깔때기와 두 눈 사이에있는 주요 두부 혈관을 폭로 맨틀 다시 가져옵니다. </li><li> 26.5 - 게이지 바늘, 바늘 두개의 혈관과 hemolymph의 50-100 μl 사이의 철회와 살균 한 ML의 주사기를 사용하여. 얼음 멸균 1.5 ML 튜브의 hemolymph를 놓습니다. 참고 : 동물 기증​​자 여러 번 역할을한다면, 오직 주어진 시간에 hemolymph의 10-20 μl를 철회. 일반 해수 탱크에 동물을 반환합니다. 동물은 30 분 이내에 회복됩니다. </li><li> 갓 수집 hemocytes는 오징어 링어의 솔루션 (; 530 MM NaCl, 10 MM KCl, MgCl 2 25 MM 10 MM CaCl 2 10 MM HEPES 버퍼, 산도 7.5 S - 링거스) 500 μl에 씻어 다시 중지됩니다. </li><li> Hemocyte 농도는 hemocytometer에 의해 결정되며, 약 2,000 세포 chambered 유리 커버 풀렸는데에 추가하고, 실온에서 10 분에 대한 유리에 준수해야 사용할 수 있습니다. 이 밀도에서는 hemocytes은 유리 슬라이드 표면에 균일 monolayer를 형성합니다. </li><li> 호스트 hemocytes에 바인딩 세균 관찰, hemocytes 그러한 장염 fischeri ES114 및 / 또는 장염 harveyi B392, 녹색 형광 기자를 포함한 각으로 찬란 표시 박테리아 변종에 노출되어 있습니다. V. fischeri ES114 및 V. harveyi B392은 28에서 바다 물이 tryptone 미디어 (SWT)의 중간 단계 로그 ° 궤도 통에 C로 성장하고 있습니다. 600nm에서 광학 밀도는 셀 밀도를 결정하기 위해 spectrophotometrically 측정됩니다. 박테리아가 원심 분리 (5 분 5000 RPM)에 의해 pelleted 있으며, 표면에 뜨는이 무시되고 펠렛은 S - 링거스에 다시 일시 중지되었습니다. </li><li> 100,000 세균성 세포가 잘하므로 평균 hemocyte 50 세균이있다는 것을 각 챔버에 추가됩니다. hemocyte / 세균 혼합물은 25 S - 링어 S 솔루션 ° 1 H에 대한 C, 바인딩의 최대 수준을 항복하기로 결정 시간 incubated 수 있습니다. </li><li> hemocytes의 세포질은 다음 찬란 0.005 % CellTracker 오렌지 (Invitrogen)와 스테인드 후 세포를 시각화하는 S - 링거스에 씻어 수 있습니다. </li><li> 관련 박테리아 물들다 hemocytes는 자이스 혈구 디스커버리 V20 형광 입체경 또는 Leica SP2 스펙트럼 레이저 공촛점 현미경을 사용하여 형광 볼, 그리고 동물 세포의 전체 표면을 통해 열거됩니다. </li></ol> 대표 결과 두 족류 동물의 hemolymph가 산소에 세포 hemocyanin 아니라 헤모글로빈을 포함하고 있기 때문에, hemolymph은 진한 파란색을 설정합니다. hemolymph의 μl 당 ~ 5000 hemocytes의 평균이 방법을 사용하여 얻을 수 있습니다. chambered 커버 풀렸는데 형광 염색법을 준수 후 hemocytes는 모양 밝은 찬란 빨간색과 amoeboid 나타납니다. 세균 유착, V에 대한 fischeri 동안 V. (혈액 세포 당 1-2 세균 세포) hemocytes을 제대로 준수합니다 harveyi는 (hemocyte 당 10-15 세균 세포) 적극 준수합니다. <img src="/files/ftp_upload/1714/1714fig1.jpg" alt="그림 1" /> 그림 1. 성인 하와이 밥테일 오징어 Euprymna은 두부 혈관의 위치를 보여주는 scolopes. <img src="/files/ftp_upload/1714/1714fig2.jpg" alt="그림 2" /> 그림 2. 장염 fischeri (A)와 장염 harveyi (B)에 hemocyte 노출 결과. 레드 셀 커 오렌지, 그린, GFP - 라벨이 세균.

<ol>
	<li>Koropatnick, T. A., Kimbell, J. R., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+host+hemocytes+during+the+initiation+of+the+squid-Vibrio+symbiosis.">Responses of host hemocytes during the initiation of the squid-Vibrio symbiosis.</a> Biol. Bull. 212, 29-39 (2007).</li><li>McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unseen+forces:+the+influence+of+bacteria+on+animal+development.">Unseen forces: the influence of bacteria on animal development.</a> Dev. Biol. 242, 1-14 (2002).</li><li>McFall-Ngai, M. J., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiont+recognition+and+subsequent+morphogenesis+as+early+events+in+an+animal-bacterial+mutualism.">Symbiont recognition and subsequent morphogenesis as early events in an animal-bacterial mutualism.</a> Science. 254, 1491-1494 (1991).</li><li>Nyholm, S. V., Stewart, J. J., Ruby, E. G., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+between+symbiotic+Vibrio+fischeri+and+the+haemocytes+of+Euprymna+scolopes.">Recognition between symbiotic Vibrio fischeri and the haemocytes of Euprymna scolopes.</a> Environ. Microbiol. 11, 483-493 (2009).</li><li>Nyholm, S. V., McFall-Ngai, M. 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+winnowing:+establishing+the+squid-Vibrio+symbiosis.">The winnowing: establishing the squid-Vibrio symbiosis.</a> Nat. Rev. Microbiol. 2, 632-642 (2004).</li><li>Nyholm, S. V., Stabb, E. V., Ruby, E. G., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+animal-bacterial+association:+recruiting+symbiotic+Vibrios+from+the+environment.">Establishment of an animal-bacterial association: recruiting symbiotic Vibrios from the environment.</a> Proc. Natl. Acad. Sci. USA. 97, 10231-10235 (2000).</li><li>Nyholm, S. V., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+the+light-organ+microenvironment+of+Euprymna+scolopes:+description+of+a+population+of+host+cells+in+association+with+the+bacterial+symbiont+Vibrio+fischeri.">Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri.</a> Biol Bull. 195, 89-97 (1998).</li><li>Visick, K. L., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrio+fischeri+and+its+host:+it+takes+two+to+tango.">Vibrio fischeri and its host: it takes two to tango.</a> Curr. Opin. Microbiol. 9, 632-638 (2006).</li></ol>

유익한 세균과 호스트 사이의 분자 신호를 중재에 면역 체계의 역할에 관한 연구, 최근에 그들의 microbiota와 eukaryotes의 공동 진화에 대한 우리의 이해에 중요한 기여를 만들었습니다. 오징어 / 비브리오 시스템은이 분야 2,3,5,6,8의 근본적 질문에 답변을 취급하기 쉬운 모델 시스템으로 자신을 증명되었습니다. 오징어 Euprymna scolopes의 가벼운 기관은 발광 박테리아 비브리오 fischeri?...

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		<th>Material Name</th>
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		<th>Comment</th>
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<td>Lab-Tek chambered #1.0 borosilicate coverglass system (8-chambers)</td>
<td>&nbsp;</td>
<td>Thermo-Fisher Scientific</td>
<td>155411</td>
<td>&nbsp;</td>
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<td>26.5 G 1ml latex-free insulin syringe</td>
<td>&nbsp;</td>
<td>Becton, Dickinson and Company</td>
<td>C34551</td>
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<td>Cell Tracker Orange</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C34551</td>
<td>&nbsp;</td>
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<td>SteREO Discovery V20 Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>SP2 Confocal Microscope</td>
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<td>Leica</td>
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obtaining hemocytes from the hawaiian bobtail squid euprymna scolopes and observing their adherence to symbiotic and non-symbiotic bacteria

유익한 세균과 호스트 사이의 분자 신호를 중재에 면역 체계의 역할에 관한 연구, 최근에 그들의 microbiota와 eukaryotes의 공동 진화에 대한 우리의 이해에 중요한 기여를 만들었습니다. 하와이 밥테일 오징어 사이의 공생 협회,<em&gt; Euprymna scolopes</em&gt; 그리고 발광 생물 세균<em&gt; 장염 fischeri</em&gt; 동물의 개발에 유익한 박테리아의 영향을 이해하기위한 모델 시스템으로 이용되었습니다. 최근 연구는 대식 세포와 같은 hemocytes은 오징어 호스트의 타고난 면역 체계의 유일한 세포 구성 요소, 가능성이 설립이 협회의 유지 보수를 중재에 중요한 역할을하는 것으로 나타났습니다. 이 프로토콜은에서 hemocytes를 얻는 방법을 보여줍니다 것입니다<em&gt; E. scolopes</em&gt; 그리고 박테리아 바인딩 assays에서 이러한 세포를 사용합니다. hemolymph가 기본 두부 혈관에서 주사기로 수집하기 전에 어른 오징어 먼저 anesthetized 있습니다. 압축을 푼 hemolymph에 포함된 호스트 hemocytes는, chambered 유리 coverslips을 준수하고 녹색 형광 단백질로 분류 공생에 노출되는<em&gt; 장염 fischeri</em&gt;이 아닌 공생<em&gt; 장염 harveyi</em&gt;. hemocytes은 형광 염료 (셀 트래커 오렌지, Invitrogen)에 counterstained 후 형광 현미경을 사용하여 시각입니다.

Watch this Scientific Journal Video about Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria at JoVE.com

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

이 동영상은 하와이 밥테일 오징어에서 hemocytes (혈액 세포)을 구하는 방법을 보여줍니다 것입니다<em&gt; Euprymna scolopes</em&gt; 세포 생물학 및 세균 유착 assays에 사용하기 위해. Hemocytes은 형광 염료와 스테인드 및 GFP - 라벨 박테리아에 노출됩니다.

하와이 밥테일 오징어에서 Hemocytes 구하기 Euprymna scolopes와 공생이 아닌 공생 박테리아들의 준수를 관찰

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

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Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

13.1K 조회수.  University of Connecticut. 이 동영상은 하와이 밥테일 오징어에서 hemocytes (혈액 세포)을 구하는 방법을 보여줍니다 것입니다 Euprymna scolopes 세포 생물학 및 세균 유착 assays에 사용하기 위해. Hemocytes은 형광 염료와 스테인드 및 GFP - 라벨 박테리아에 노출됩니다.

비디오: Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

Xenopus laevis 여성의 난자를 구하기

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

연구

생물학

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

세포 생존에 박테리아와 그 영향의 동결 건조 공법

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

야생 유충에서 Hemocytes을 추출하기위한 간단한 프로토콜

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between 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 ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

오징어로부터 색소를 추출하는 방법<em&gt; Doryteuthis의 pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

녹색 형광 단백질 박테리아에서 감염 하는 동안 전달 하는 이펙터를 시각화 하기 위해 시스템을 분하시오

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 &#181;m filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.

We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

구강에서 공생 사카리박테리아의 안정적인 이진 문화 구축

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

대장균 및 다른 박테리아로부터의 세포외 소포의 확장 가능한 분리 및 정제

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...