이 작품은 DFG에 의해 자금입니다.

비 준비 및 백 - 채우기 <ol><li> 고정을위한 얼음 하이브와 감기에 꿀 비 foragers을 관람해보세요. </li><li> 플렉시가 실 3 녹화에 탑재합니다. 낮은 융점 하드 왁스로 녹음 챔버 벽에 눈과 흉부을 수정. </li><li> 일반적으로 전기 생리학 사용으로 유리 모세관을 당겨 및 CA의 팁 직경을 얻는 끝에 휴식. 10μm. 염색 붙여있는 모세관의 끝부분을 커버. 다이 붙여넣기 Fura - 2 dextran과 리신 고칠수 tetramethylrhodamine dextran의 10시 1분 혼합물로 구성되어 있습니다. </li><li> 얼룩 절차에 대한 세밀한 점 또는 N - eicosane와 안테나를 무력화. 두뇌 위의 표피의 일부를 제거하고 버섯 본문에 대한 액세스를 허용하는쪽으로 분비 및 기관 밀어. </li><li> 소마 또는 버섯 신체 뉴런의 axonal 지역 중 하나에 모세 혈관을 주입. 이것은 무료로 손으로 일을하거나 마이크로 조작하는 사람을 사용하실 수 있습니다. 그런 다음 머리를 캡슐에 표피 조각을 복원하고 안테나를 푸세요. </li><li> 20 적어도 4 시간 동안 humidified 경우에 그들을 저장하기 전에 야간 이상 30 % 자당 솔루션 꿀벌 먹이 ° C. </li></ol> 생체내 이미징에 <ol><li> 복부에 대한 눌렀을 및 기록 챔버에 클립이나 테이프로 고정 스펀지의 작은 조각으로 비 예의 움직임을 방지합니다. </li><li> 식도의 펌핑으로 인한 두뇌의 움직임을 방지하기 위해, labrum 위의 표피에 작은 절개를 잘라 조심스럽게 식도를 꺼내와 그 주변 단단한 구조 식도 4 손상없이 긴장 밑에 넣고. 이 구성 요소의 실리콘과 커버. </li><li> 바늘로 구멍을 만들어 M17의 녹음 와이어 전극을 삽입합니다. </li><li> 표피 조각, 기관 및 뇌 이상 분비를 제거합니다. 종이로 헤드 캡슐 내부 heamolymph 석. </li><li> 이 구성 요소의 실리콘으로 헤드 캡슐을 입력합니다. 두뇌가 완전히 덮여 것이 중요합니다. </li><li> 플레이스 현미경 스테이지에서 꿀벌과 비말에 현미경의 딥 목적을 담가하는 실리콘의 표면에 물 한 방울을 넣으십시오. 스테인드 뉴런에 중점을두고 있습니다. </li></ol> 신호의 냄새 자극 및 녹화 Olfactometer : 우리는 컴퓨터를 제어, 사용자 지정 구축 자극 장치 또는 Galizia와 베터 3 이전에 설명한 것처럼 &quot;olfactometer&quot;를 사용합니다. 냄새는 안테나 향해 감독 일정한 공기 흐름으로 희석하고 있습니다. 모든 자극은 0.2ml 냄새 포화 공기의 3S로 냄새 펄스로 구성되어 있습니다. 자극 프로토콜은 레코딩 소프트웨어 TILLVision에서 설정할 수 있습니다. 설정 현미경 및 이미징 : 우리는 자이스 혈구 형광 현미경을 사용합니다. 이미지는 25 ° 현미경에 장착된까지 - Photonics 이미징 장치를 사용하여 5 Hz의 샘플링 속도와 C로 기록됩니다. 칼슘 신호는 X60, 0.9 W 올림푸스 성충 CCD 카메라 (640x480에서 픽셀, 160X120 픽셀 칩 binned 4X)와 함께 수영을 객관적으로 기록됩니다. Fura - 2는 단색 340의 빛과 ratiometric 측정 파장을 380nm로 흥분합니다. 형광은 410nm 이색성 거울과 440nm 긴 통과 필터를 통해 감지됩니다. 기록에 대한 매개 변수는 레코딩 소프트웨어에서 설정합니다. 각 측정은 두 개의 파장에 대해 10 초 및 노출 시간 각 준비에 얼룩 강도 조정될 수 있습니다 지속. M17 녹음 : labium (M17)의 오래 끄는 근육이 학습과 관련된 행동 반응을 모니터하는 extracellularly 기록됩니다 PER 5 6 즉. 입 부분에 가까운 근육에 구리 철사를 삽입. 눈에 접지 전극을 삽입. 근육 잠재력은 사용자 지정 빌드 사전 증폭기로 증폭된 디지털 및 컴퓨터에 저장됩니다. 자극 증상은 olfactometer에 의해 트리거됩니다. 데이터 분석 <ol><li> 실험을 진행하는 동안 : 칼슘 신호의 녹음은 컴퓨터에 공급하고 저장됩니다. 레코딩 소프트웨어 TILLVision의 파장 (340nm/380nm)와 deltaF (기준의 뺄셈)의 계산 간의 비율 계산 같은 신호의 초기 검사를하실 수 있습니다. </li><li> 이미지 처리는 IDL로 작성된 사용자 정의 프로그램이 수행됩니다. 칼슘 2 비율 + 340nm에서 신호를 각 픽셀에 대한 380nm 측정을 계산합니다. 자극 전에 프레임을 통해 평균하여 형광 배경 (F)을 확인하고 ratiometric 신호 (deltaF)에서 빼야. </li><li> 관심 (ROI)의 지역을 정의하려면 다음과 같이 자극하는 동안 프레임의 의미와 저역 통과 필터 (3X3px)를 적용 계산합니다. 잘못된 색상 규모에 그레이 스케일로 변환. 거짓 컬러 이미지에서 활동 명소로의 연결 구조 활동을 파악하고 투자 수익 (ROI)로 정의합니다. </li><li> 어떤 필터링이나 수정없이 프레임 이상의 투자 수익 (ROI)의 픽셀을 평균하여 활성 영역의 관자놀이 역학을 계산합니다. </li></ol> 함께 Fura - 2 dextran과 함께 우리는 고칠수 염료 rhodamine dextran ( &quot;미니 루비&quot;)과 뉴런을 입력합니다. 두 염료 같은 분자량을 가지고, 그들은 뉴런에 공동 위치해야합니다. 와이드 필드 영상은 매우 제한된 공간 해상도를 허용, 따라서 그것은 공촛점 스캐닝 현미경을 사용하여 실험 후 스테인드 구조를 조사하는 것이 좋습니다. <ol><li> 생리적 측정 후, 머리를 해부하다하고 흥분시키는. </li><li> PBS 및 에탄올과 메틸 salycilate의 혐의 stepwise 탈수에 씻어. </li><li> 공촛점 현미경에 대한 메틸 salycilate의 머리를 포함. </li><li> 우리는 Leica TCS SP2 현미경을 사용합니다. 여기 파장은 543nm이다. </li><li> 우리는 공기 또는 기름 침지 목적으로 두뇌를 스캔하고 이미지 데이터와 스테인드 구조를 비교합니다. </li></ol> 대표 결과 : 얼룩과 준비가 성공했다면, 허위 색상 규모의 신호, 버섯 신체 알파 엽 외부 뉴런에서 예를 들어, 그림 1처럼 보이게 수 있습니다. 행동 응답 (당) 자극 (figure2) 전에 자극 마이너스 스파이크 주파수 중에 스파이크 주파수로 계산하실 수 있습니다. 스테인드 구조는 공촛점 현미경 (figure3)를 사용하여보다 상세히 조사 수 있습니다. <img src="/files/ftp_upload/1353/figure1_new.png" alt="그림 1" /> 그림 1 : 대표 칼슘 이미징 신호 (heptanal와 자극). A : 비 뇌의 몇 군데 지역. B : 380nm 여기 파장에서 X20 물놀이를 객관적으로 볼 수로 원료 형광 이미지, 버섯 본문의 알파 - 엽은 자주색으로 설명되어 있습니다. 염료는 불순물 뉴런을 얼룩이 알파 엽 (叶)의 ventro - 측면 가장자리에 주입했다. C : 삼초 자극하는 동안의 평균 신호 deltaF (380분의 340)에서 파생된 거짓 색상 이​​미지, 자주색에 명시된 알파 엽 선택한 투자 수익 (ROI)은 검정색으로 설명했다. D : 냄새 자극, C. E의 표시로 관심 지역에서 평균 신호에 대한 응답으로 칼슘 신호의 시간적 역학 : 투자 수익 (ROI)에 각각 자당의의 자극 ipsi과 콘트라 측면 안테나에 대한 응답으로 칼슘 신호의 시간적 역학 표시 C로 <img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> 그림 2 : 대표 M17 응답. 후각 컨디셔닝 후 꿀벌 그 냄새가 보상없이 제시 코를 확장합니다. 레드 라인 냄새 자극을 나타냅니다. 삽입된 페이지 : 꿀벌의 PER. <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> 그림 3 : 대표 이미지 스택. 이미징 실험 후 머리가 해부했다. 그림 1에서 신경 세포는 보라색에 명시된 X20 오일 침지 목표, 알파 - 엽과 공촛점 현미경을 사용하여 스캔합니다.

<ol>
	<li>Joerges, J., K&#252;ttner, A., Galizia, C. G., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representations+of+odors+and+odor+mixtures+visualized+in+the+honeybee+brain.">Representations of odors and odor mixtures visualized in the honeybee brain.</a> Nature. 387 (6630), 285-288 (1997).</li><li>Rehder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+honeybee's+proboscis+reflex+by+electromyiographic+recordings.">Quantification of the honeybee's proboscis reflex by electromyiographic recordings.</a> J. Insect Physiol. 33, 501-507 (1987).</li><li>Galizia, C. G., Vetter, R. S., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Methods+for+Analyzing+Odor-Evoked+Activity+in+the+Insect+Brain.">Optical Methods for Analyzing Odor-Evoked Activity in the Insect Brain.</a> Methods in Insect Sensory Neuroscience. , 345-388 (2004).</li><li>Mauelshagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+correlates+of+olfactory+learning+paradigms+in+an+identified+neuron+in+the+honeybee+brain.">Neural correlates of olfactory learning paradigms in an identified neuron in the honeybee brain.</a> J Neurophysiol. 69 (2), 609-625 (1993).</li><li>Kuwabara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bildung+des+bedingten+Reflexes+von+Pavlovs+Typus+bei+der+Honigbiene+Apis+mellifica,+Hokaido+Univ.">Bildung des bedingten Reflexes von Pavlovs Typus bei der Honigbiene Apis mellifica, Hokaido Univ.</a> Zool. J. Sci. 13, 458-464 (1957).</li><li>Bitterman, M. E., Menzel, R., Fietz, A., Schafer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+of+proboscis+extension+in+honeybees+(Apis+mellifera.">Classical conditioning of proboscis extension in honeybees (Apis mellifera.</a> Journal of Comparative Psychology. 97 (2), 107-119 (1983).</li></ol>

이 프레 젠 테이션에서 우리는 꿀벌의 생체내 칼슘 신호에 조사하기위한 모든 단계를 통과했습니다. 우리는 버섯 몸의 신경이 기술을 적용하지만, 이미지는 얼룩 기술이 확립 수있는 모든 뉴런에서 할 수 있습니다. 우리는 벌꿀 꿀벌의 후각 경로의 뉴런에 주력했습니다. 실험은 모든 암실에서 수행됩니다. 세트는 최대 꿀벌의 광학 인식 범위 밖으로 거짓과 염료를 자극하지 않는 긴 ...

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<tr>
<td>low melting point hard wax Deiberit 502</td>
<td>&nbsp;</td>
<td>Dr. B&ouml;hme & Sch&ouml;ps Dental GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FURA-2 dextran potassium salt, 10 000 MW</td>
<td>&nbsp;</td>
<td>Invitrogen/Molecular Probes</td>
<td>F-3029</td>
<td>Protect from light.</td>
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<tr>
<td>tetramethylrhodamine dextran 10 000 MW</td>
<td>&nbsp;</td>
<td>Invitrogen/Molecular Probes</td>
<td>D-3312</td>
<td>Protect from light.</td>
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<tr>
<td>n-eicosane</td>
<td>&nbsp;</td>
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<td>WPI</td>
<td>KWIK SIL</td>
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<td>TILL Photonics</td>
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<td>Imago</td>
<td>&nbsp;</td>
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<td>CED</td>
<td>&nbsp;</td>
<td>Texas Instruments</td>
<td>&nbsp;</td>
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in vivo ca2+- imaging of mushroom body neurons during olfactory learning in the honey bee

생체내 및 반에서 칼슘 이미징에 대한 생체내 준비가 Joerges, Küttner 및 Galizia에 의해 우리가 실험실에서 개발되었습니다의 이상 십년 전, antennal 엽 1 냄새 evoked 활동을 측정합니다. 그때부터, 그것은 지속적으로 세련되었고 비 두뇌에서 다른 neuropiles에 적용. 여기서 우리는 현재 dextran 결합 칼슘에 민감한 염료를 (Fura - 2)를 사용하여 버섯 신체 뉴런의 활동을 측정하기 위하여 실험실에서 사용되는 준비를 설명합니다. 우리는 retrogradely 자신의 axons 또는 소마 지역에 염료를 주입하여 버섯 신체 뉴런을 얼룩. 우리는 에어컨 PER 사용하여 꿀벌을 양성 볼 수 있습니다있는 준비를 달성하기 위해, invasiveness을 줄이는 데 중점을 둡니다. 우리는 PER (M17) 2를 제어하는 근육에서 전자 myograms을 기록하여 행동 반응을 모니터링하고 정할 수 있습니다. 생리 실험 후 몇 군데 구조는 뉴런의 신원을 주소로 공촛점 스캐닝 현미경을 사용하여보다 상세히 조사하고 있습니다.

Watch this Scientific Journal Video about In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee at JoVE.com

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

꿀벌은 appetitive 후각 학습 패러다임 (당 시설)에서 시설 수 있습니다. 자극과 같은 냄새를 사용하여, 우리는 동시에 칼슘 이미징이 버섯 신체 뉴런에서 냄새 evoked 활동을 측정하는 데 사용하는 동안 기록되는 행동 방식을 설립<em&gt; 생체내에</em&gt;.

생체내 칼슘 2 + - 허니 비에 강한 학습하는 동안 버섯 바디 뉴런의 영상

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

in-vivo-ca2-imaging-mushroom-body-neurons-during-olfactory-learning

Research

JoVE Journal

Biology

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

13.9K 조회수.  Freie Universität Berlin. 꿀벌은 appetitive 후각 학습 패러다임 (당 시설)에서 시설 수 있습니다. 자극과 같은 냄새를 사용하여, 우리는 동시에 칼슘 이미징이 버섯 신체 뉴런에서 냄새 evoked 활동을 측정하는 데 사용하는 동안 기록되는 행동 방식을 설립 생체내에.

비디오: In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

성인 마우스의 후각 행동 테스트

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

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Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

만성에 대한 마이크로 드라이브 배열 생체내에 녹화 : 드라이브 제작

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential amplitude of action potential signatures over the channels of a tetrode allows for the isolation of single-unit activity from multi-unit signals.  The ability to precisely control the stereotaxic location and depth of the tetrode is critical for studying coordinated neural activity across brain regions.  In combination with a micro-drive array, it is possible to achieve precise placement and stable control of many tetrodes over the course of days to weeks.  In this protocol, we demonstrate how to fabricate and condition tetrodes using basic tools and materials, install the tetrodes into a multi-drive tetrode array for chronic in-vivo recording in the rat, make ground wire connections to the micro-drive array, and attach a protective cone onto the micro-drive array in order to protect the tetrodes from physical contact with the environment.



Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

만성에 대한 마이크로 드라이브 배열 생체내에 녹화 : Tetrode 조립

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

이미징 CA의 기술 2 + 인간 정자의 시그널링

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

헤어 재생시 상피 - 중간 엽 시그널링의 빠른 유전 분석

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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

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

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

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

둔화 가속과 반전 꿀 꿀벌 모델에서 노화와 표본을 얻기

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

아프리칸 유럽 꿀 꿀벌에 의해 만들어 Beebread의 영양소를 비교하는 방법과하는 Hemolymph 단백질 역가에 미치는 영향

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

이미징 로컬 CA<sup&gt; 2 +</sup&gt; 배양 된 포유 동물 세포에서 신호

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...