이 작품은 CNPq (게 나시오날 드 Desenvolvimento Científico e Tecnológico), 망 (Coordenação de Aperfeiçoamento 데 피해 드 높이 등)에 의해 지원 되었다, FAPERN (Fundação de Amparo 검색 할 리오 그란데 노르 테 할).

All experiments involving live animals described in this protocol are conducted according to the National and International laws and were approved by the local University Animal Care and Use Committee (CEUA/UFRN), under the license 009/2014. The following protocol is performed in a sterile environment. Familiarity with basic cell culture is expected.
1. Dorsolateral Telencephalon Microdissection
<ol>
	<li>Prepare the dissection medium (100 mL): 98.5 mL Hank&#39;s Balanced Salt Solution (HBSS), 0.5 mL 5 M HEPES pH 7.4, 1 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL).</li>
	<li>Filter-sterilize the dissection medium.</li>
	<li>Remove embryonic day 14 (E14) embryos from a pregnant mouse C57/Bl6 (Mus musculus) under anesthesia with isoflurane. 
	NOTE: Consider E0 as the day of vaginal plug detection. Dissection should be completed no later than 1 h after removal from the pregnant mouse. This protocol also applies for embryos E11-E17.</li>
	<li>Remove the embryos by hysterectomy under sterile conditions.</li>
	<li>Transfer the embryos to a Petri dish with cold dissection medium (4 &#176;C).</li>
	<li>Remove the brains from the skull by cutting along the longitudinal fissure. The number of embryos usually varies from five to ten. Each E14 brain yields approximately 2 x 106 cells, including progenitors and postmitotic neurons (1:1 ratio) 
	NOTE: Use the stereomicroscope for the following steps.</li>
	<li>Remove the meninges using dissection forceps. Split the telencephalon in the middle to separate the hemispheres. To isolate the dorsolateral telencephalon, cut along the dorsomedial curve and pallial-subpallial boundary using dissection forceps. Transfer the dorsolateral telencephalon to a 2 mL tube with cold dissection medium (4 &#176;C) until all brains are dissected.</li>
</ol>
2. Cell Dissociation and Plating
<ol>
	<li>Prepare the proliferation medium (50 mL of DMEM+10% FCS): 44 mL Dulbecco Modified Eagle&#39;s Medium (DMEM), 5mL Fetal Calf Serum (FCS), 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL). Filter-sterilize the proliferation medium.</li>
	<li>Prepare differentiation medium (50 mL of DMEM+2% B27): 48 mL DMEM, 1 mL B27, 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL). Filter-sterilize the differentiation medium.</li>
	<li>After micro-dissection of the dorsolateral telencephalon from E14 mice, centrifuge the tube with the collected tissue and cold dissection medium (for 5 min at 4 &#176;C, 340 x g) to precipitate the tissue.</li>
	<li>Remove the supernatant with a pipette and add 1 mL of pre-warmed (37 &#176;C) Trypsin-EDTA (0.05%) for chemical digestion. Incubate for 15 min at 37 &#176;C. Add 2 mL of proliferation medium to stop trypsin activity.</li>
	<li>Polish the tip of a glass Pasteur pipette over a gas burner or Bunsen burner for few seconds to slightly narrow the aperture. Wash the pipette by aspirating FCS to coat the tip. Avoiding bubbles, dissociate mechanically the cells with a fire-polished and FCS-coated Pasteur pipette.</li>
	<li>Centrifuge the cells (for 5 min at 4 &#176;C, 340 x g). Remove supernatant with a pipette. Add 1 mL of proliferation medium and resuspend the cells using a pipette. Repeat this step once.</li>
	<li>Prepare a 1:1 dilution of the cell suspension using a 0.4% Trypan Blue solution. Non-viable cells will be blue. Count the number of viable cells (unstained) using a Neubauer chamber.</li>
	<li>Dilute the cells in proliferation medium to get 106 cells/mL. Add 500 &#181;L of the cell suspension to each well of a 24 well tissue culture plate (approximately 2.5 &#215; 105&#160;cells/cm2). Incubate cells at 37 &#176;C and 5% CO2. 
	NOTE: Do not use glass coverslips, as they can move during the experiment and change the field of observation. Alternatively, use glass bottom tissue culture plates. 
	NOTE: If you are not using cell-culture treated multidished, it is important to pre-treat your plates with Poly-D-Lysin (50 &#181;g/ml) 2 h at 37 &#176;C. PDL-treated plates can be stored at 4 &#176;C after washing with distilled water.</li>
</ol>
3. Retroviral-mediated Transfection
NOTE: This is a simple method to transfect only progenitor cells. However, other viral vectors or chemical/electrical transfection can be used to insert genes of interest in cultured cells.
<ol>
	<li>To manipulate gene expression and label cells with fluorescent reporter proteins, add retroviruses carrying plasmids with genes of interest 2 h after cell plating. 
	NOTE: Retroviral vectors only integrate into the genome of dividing cells. Therefore, it is important to infect cells when proliferation is high. 
	NOTE: We used a plasmid containing the Beta-actin/CMV fusion sequence (CAG) and an internal ribosomal entry site (IRES) followed by the coding sequence for the green fluorescent protein (GFP), designated pCAG-IRES-GFP44. Genes of interest could be cloned between the CAG promoter and IRES sequence37.</li>
	<li>Dilute the retrovirus in differentiation medium to obtain the desired number of particles per mL. Add 500 &#181;L of differentiation medium with retrovirus to each well. 
	NOTE: The amount of retroviruses used is dependent on its titer and should be calculated to obtain 50 to 100 infected cells per well. This titer allows the identification of isolated GFP-expressing clones, whose relationship can be further confirmed by tracking phase contrast images (see section 6). The titration of retrovirus should be performed using the same cell population45.</li>
</ol>
4. Time-lapse Video-microscopy
NOTE: This step requires an inverted fluorescence microscope with incubation chamber (see Table of Materials).
<ol>
	<li>After the retrovirus infection, place the tissue culture plate into an incubator connected to temperature and CO2 controllers. 
	NOTE: Preheating the chamber for 3 h prior to starting imaging could help minimize drift and defocus during the acquisition. 
	NOTE: The chamber should maintain the cells at constant conditions of 37 &#176;C and 5% CO2.</li>
	<li>Select a position in the XY axis to serve as a zero-point (XY = 0). This will allow a reference to find the positions again in the future, for instance after post-imaging immunocytochemistry (see section 5). 
	NOTE: It is advisable to draw a sign in the plate that can be used as a reference to determine the zero-point.</li>
	<li>Select the positions to be imaged at 10X magnification. 
	NOTE: Select 10-15 positions per well to increase the likelihood to observe retrovirally-transfected cells. 
	NOTE: Long-distance high magnification objectives (20X or 40X) can be used to gain higher resolution images. Depending on the working distance of objectives, glass-bottom plates may be required.</li>
	<li>Acquire phase contrast images every 5 min and fluorescence images every 3 h to decrease phototoxicity. Check and adjust the focus in the first 3 h of experiment, as it may change while the temperature equilibrates. Check and adjust the focus daily. 
	NOTE: We observe that the focus becomes stable after the first 24 h. However, it is advisable to monitor the focus.&#160;Alternatively, use a software-based autofocus system.</li>
	<li>After 7 days, stop acquisition and proceed to post-imaging immunocytochemistry (see section 5).</li>
	<li>After immunocytochemistry, replace the plate in the microscope incubator, find the zero-point, reset XYZ = 0 and reload the experiment with saved positions. Acquire the fluorescence images of each position using appropriate fluorescence filters.</li>
</ol>
5. Post-imaging Immunocytochemistry
<ol>
	<li>After 7 days in culture, fix the cell culture with 4% Paraformaldehyde (PFA) fixative for 15 min at room temperature. 
	CAUTION: PFA is toxic and should be handle in a chemical fume hood.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>Incubate in primary antibodies overnight at 4 &#176;C in 0.5% triton and 10% of normal goat serum in PBS 0.1 M. 
	NOTE: Use anti-Microtubule Associated Protein 2 (MAP2) antibody to confirm the neuronal phenotype (mouse IgG1, 1:1,000) and anti-GFP to label the transduced cells (chicken antibody, 1:500). Other antibodies can be used to detect progenitor or glial cells.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>Dilute secondary antibodies in 0.5% triton and 10% of normal goat serum in PBS 0.1 M and incubate for 2 h at room temperature. Recommended dilution: 1:1,000.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>For nuclei staining, incubate the cells for 5 min with 0.1 &#181;g/mL 4&#8242;6&#8242;-diamino-2-phenylindone (DAPI) in PBS 0.1 M.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>Keep the cells in PBS 0.1 M and protect from light (e.g., wrap the plate with foil).</li>
</ol>
6. Cell Tracking
NOTE: Here, we briefly describe the main steps to analyze time-lapse video-microscopy data using the software The Tracking Tool (tTt), which is available at the link provided in Table of Materials.
NOTE: If the image acquisition is performed with other software, import the image data with tTt Converter available in the tTt installer (link provided in Table of Materials). Only png, tif or jpg image formats are accepted. Follow the steps shown at Figure 1 A - B after starting the tTt Converter.
<ol>
	<li>Start the software tTt and choose a user name. Click on &#34;add user&#34; and &#34;continue&#34; button (Figure 2A). Browse and select the &#34;TTTWorkFolder&#34; (Figure 2B). Lineages trees, statistics and exported images will be saved in this folder. Next, select the experiment to be analyzed and load it (Figure 2B).</li>
	<li>If the images were converted previously by tTt Converter, click on &#34;LogFileConverter&#34; (Figure 3A) and specify the number of seconds between two consecutive time points in the LogFileConverter window. Click on &#34;Convert log files for whole experiment&#34; (Figure 1C).</li>
	<li>In a list of images, which appear in the tTt window, select the desired images that by loading manually or using the option displayed in the program. Load images (Figure 3).</li>
	<li>Select File &#62; Open &#62; New colony. Click on &#34;Tracking&#34;, followed by &#34;Start Tracking&#34; to start the tracking in a chosen position.</li>
	<li>A movie window will appear (Figure 4). Select the cell using the tracking circle and press the key 0. The software will go to the next frame. Keep on positioning the tracking circle around the tracked cell using the mouse and click 0 in each frame to add a track mark on the cell.</li>
	<li>Use the keys 1 and 3 to move to the previous or next frame, respectively. To delete a track mark, just click on the correct position to mark and press 0.</li>
	<li>If the cell divided, select the button &#34;division&#34; (red rectangle at Figure 4). Continue tracking one daughter cell by pressing Shift+D. To track the second daughter cell, select it in the cell editor and press F2.</li>
	<li>If the cell died during the video microscopy, select &#34;apoptosis&#34; (blue rectangle at Figure 4). 
	NOTE: Although the software labels the event as &#34;apoptosis&#34;, other mechanisms may be responsible for the cell death.</li>
	<li>If the cell leaves the field of observation or intermingles with neighboring cells precluding tracking, select &#34;lost&#34; (green rectangle at Figure 4).</li>
	<li>To stop tracking and continue later click on &#34;interrupt&#34; (purple rectangle at Figure 4). 
	NOTE: In the Cell editor window a tree will be generated during the tracking. To save the lineage tree, select File &#62; Save &#62; Current tree (in the Cell editor window).</li>
	<li>To start tracking a different cell, click on the cell using the right mouse button or select the cell using the left mouse button and click on Tracking &#62; Start Tracking.</li>
	<li>To export the movie with the tracking data, the software should not be in &#34;tracking mode&#34;. To turn off the tracking mode, execute step 6.10. In the Movie window select Export &#62; Movie. Set up the format, range of frames, number of frames per second and bitrate. Click on &#34;Start export&#34;.</li>
</ol>
7. Quantifications
NOTE: Several measurements can be performed using video microscopy data46. Here, we describe three possibilities that are exemplified later in the &#34;representative results&#34; section.
<ol>
	<li>Quantify the cell survival for each cell lineage by dividing the number of cells alive at different time points by the total number of cells generated before these time points were reached within individual clones37.</li>
	<li>Quantify the proportion of symmetric progenitor (SP, both daughter cells continue to proliferate), asymmetric (A, one daughter cell continues to proliferate and the other becomes postmitotic), or symmetric terminal (ST, both daughter cells become postmitotic)15,38.</li>
	<li>Measure the cell cycle length by calculating the time span of proliferating cells between their generation and division15.</li>
</ol>

저자는 공개 없다.

실시간으로 관찰 기본 대뇌 피 질 세포의 세포 증식의 분석, 세포 분열, 세포 주기 길이, 세포 분화 및 세포 생존3,14,,1537의 모드 수 있습니다. 더 중요 한 것은, 그것은 신경3NSCs에서 진행 하는 동안 제정 중간 단계의 식별으로 이어지는 단일 세포 계보의 연구 허용. 마지막으로, 공학 ?...

신경 줄기 세포 (NSC)는 대뇌 피 질 개발 중 신경 및 macroglial 셀을 생성합니다. 초기-corticogenesis, NSCs 대칭 세포 분열의 여러 라운드를 받 다 고 뿌리 풀 확장. 다음, NSCs 나누기 비대칭 중간체1를 통해 직접 또는 간접적으로 뉴런을 생성 합니다. 에 중반-에 늦은-corticogenesis, 창시자 이다 및 oligodendrocytes2,,34생성 하 전환 합니다. 그러나, 운명 제한 창시자 독특한 종류의 신경 또는 macroglial 세포의 생성에 기여 뿐만 아니라 세포 증식 및 분화, 제어 하는 완전 한 메커니즘 강렬한 토론4,,56의 문제가 남아 있습니다. 광범위 하 게 공부 생체 외에서 그리고 vivo에서 와 같은 수많은 기법을 사용 하 여 신경, 이다 oligodendrocytes를 생성 하기 위해 개별 외피 NSCs의 잠재력 되었습니다: 단일 셀 문화7,8,9,10,11,,1213; 이미징 라이브 고밀도 문화 문화3,,1415; 이미징 라이브 라이브 이미징 슬라이스 문화16,,1718; 바이러스 성 벡터 중재 유전 라벨 고밀도 문화14,15,19,,2021;를 사용 하 여 클론 분석 클론 분석에 비보에 레트로 바이러스22,23,,2425,26,27,28,29,30,31,32,33;를 사용 하 여 그리고 클론 분석에 vivo에서 유전자 변형 동물34를 사용 하 여.
이러한 기술을 각각 장점과 단점을 제공합니다. 예를 들어, 혈통 추적 vivo에서 총괄 한 그리고 분할 오류3, 개별 외피 창시자의 잠재력에 대 한 상반 된 결론을 이끌어을 받기 쉽습니다. 또한, 둘 다 생체 외에서 그리고 vivo에서 학문 초기 시간 포인트에서 조상 세포의 라벨에 따라 그리고 후부 분석 세포 계보의 혈통-진행35동안 세포 죽음의 들 키 지 않고 발생에 의해 영향을 수 있습니다. 따라서, 단일 NSCs의 잠재력 분석 하기 적합 한 시스템 생성 하는 모든 셀의 식별 뿐만 아니라 혈통 내에서 세포 운명의 적절 한 묘사를 허용 해야 합니다. 1 차 세포 배양 및 라이브 이미징의 조합에는이 설정을 제공합니다. 단일 세포 배양 및 시간 경과 비디오 현미경을 사용 하 여, 사원 외. 스위치에에서 나타났습니다 gliogenesis11성체에서 개별 대뇌 피 질 창시자의 계보. 나중에, 그들은 뉴런의 종류는 단일 외피 조상12에서 생성 됩니다 보여 동일한 시스템을 사용. 그러나,이 시스템 중요 한 경고 표시: 4 이상의 자손9의 클론을 생성 하는 이른 corticogenesis에서 절연 외피 창시자의 1%만. FGF2 추가 후 4 명 이상 세포 생성 세포의 주파수 8-109증가 합니다. 그럼에도 불구 하 고,이 숫자는 너무 작은이 단계에서 거의 모든 대뇌 피 질의 창시자는 증식을 고려. 또한, 운명 사양에 FGF2의 잠재적인 효과36배제할 수 없다. 고밀도 세포 배양 (Pax6 표현) 심 실 둘 다의 확산을 지 원하는 사용이 한계를 우회 하 고 subventricular (Tbr2 표현) 대뇌 피 질의 창시자15. 또한, 이러한 문화의 실시간 관찰 NSC 혈통 진행의 여러 가지 기능 세포 분열, 세포 주기 길어, 잠재력 단일 셀의 신경 및 명과, 다른 사람의 사이에서 생성 하는 모드 등이 조건 하에서3,15재현은 보이고 있다. 최근에, 우리는 또한 사용이 시스템 그 쥐37에서 미 성숙한 대뇌 피 질 뉴런의 세포 생존에 영향을 미치는 CREB 신호 보여. 따라서, 우리가 믿는 그 시간 경과 비디오 현미경 기본 murine 대뇌 피 질의 고밀도에 성장 하는 세포는 세포 주기 진행의 세포 및 분자 메커니즘, 세포 분열, 세포 생존 및 세포 운명 명세 모드를 공부 하는 강력 하 고 액세스할 수 도구. 후자는 수행할 수 있습니다 실시간38,,3940 에 특정 세포 운명의 식별 또는 immunocytochemistry3,38,,4142후 이미징의 사용을 허용 하는 유전자 변형 동물을 사용 하 여.
여기, 우리는 기본 대뇌 피 질 세포 배양 지원 NSCs의 확산 및 신경 및 macroglial 세포의 다음 세대를 준비 하는 단계별 프로토콜을 제공 합니다. 우리는 또한 식별 시간 경과 비디오 현미경을 사용 하 여 단일 셀 수준에서 추적 될 수 있는 개별 세포의 유전자 발현 조작 transfection retroviral 중재의 사용 설명. 설치류에 corticogenesis의 끝에 처음부터 격리 기본 대뇌 피 질 세포를 공부 하이 프로토콜을 사용할 수 있지만 몇 가지 조정 단계14에 따라 필요할 수 있습니다. 다른 소스에서 분리 하는 NSCs 또한 공부 될 수 있다 2D 문화의 시간 경과 비디오 현미경을 사용 하 여 하지만 적절 한 자란 시스템 셀 동작 생체 외에서 그리고 vivo에서38,43를 비교 하 여 결정 되어야 한다.

<table><tbody><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Invitrogen Life Technologies</td><td>14175129</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375-25G</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Dulbecco Modified Eagle&#039;s Medium (DMEM)</td><td>Gibco</td><td>12400-024</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>Glucose</td><td>Gibco</td><td>A2494001</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>trypsin-EDTA (0.05%)</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>16005</td><td></td></tr><tr><td>Goat serum</td><td>Sigma-Aldrich</td><td>69023</td><td></td></tr><tr><td>Triton X-100</td><td>VWR International Ltd.</td><td>306324N</td><td></td></tr><tr><td>Isoflurane</td><td>Sigma</td><td>792632</td><td></td></tr><tr><td>anti-MAP2, mouse</td><td>Sigma</td><td>M4403</td><td></td></tr><tr><td>anti-GFP chicken</td><td>Aves</td><td>0511FP12</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>Goat anti-mouse alexa 594</td><td>Invitrogen</td><td>A11005</td><td></td></tr><tr><td>Goat anti-chicken alexa 488</td><td>Invitrogen</td><td>A11039</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr><tr><td>tTt</td><td>ETH Zurich</td><td></td><td></td></tr><tr><td>Cell observer microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Pasteur pipette</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>The Tracking Tool (tTt) software</td><td></td><td>https://www.bsse.ethz.ch/csd/software/ttt-and-qtfy.html</td><td>download link</td></tr></tbody></table>

live imaging of primary cerebral cortex cells using a 2d culture system

대뇌 피 질 개발, 조상 세포 새로운 창시자 또는 postmitotic 신경 생성 하 대칭 및 비대칭 세포 분열의 여러 라운드를 받 다. 나중에, 일부 창시자 gliogenic 운명에 사이토와 oligodendrocyte 인구에 추가 전환 합니다. 기본 대뇌 피 질 세포 배양의 시간 경과 비디오 현미경을 사용 하 여, 세포 분열의 모드와 조상 세포의 세포 주기 매개 변수 제어 세포 및 분자 메커니즘 연구 가능 하다. 마찬가지로, postmitotic 세포의 운명은 셀 전용 형광 기자 단백질을 사용 하 여 또는 포스트 immunocytochemistry 이미징 시험 될 수 있다. 더 중요 한 것은, 이러한 기능을 모든 종류의 특정 세포 생성 하기 위해 노력 하는 창시자의 식별 수 있도록 단일 세포 수준에서 분석할 수 있습니다. 유전자 발현의 조작 또한 바이러스 중재 transfection, 세포 자치 및 셀을 자치 현상의 연구를 수를 사용 하 여 수행할 수 있습니다. 마지막으로, 퓨전 형광 단백질의 사용 부문 및 딸 세포 운명과 상관 하는 동안 선택 된 단백질의 대칭 및 비대칭 분포의 연구를 수 있습니다. 여기, 우리는 며칠에 대 한 기본 대뇌 피 질 murine 셀을 이미지 하 여 세포 분열, 세포 주기 길이 및 새로 생성 된 셀의 운명의 모드 분석 시간 경과 비디오 현미경 방법 설명. 우리는 또한 transfect 조상 세포의 유전자를 조작 하거나 단순히 기자 단백질 셀 라벨에 적용할 수 있는 간단한 방법을 설명 합니다.

Primary cultures of cerebral cortex cells isolated from embryos E14 contain both progenitor and neuronal cells. During the period of imaging, progenitors undergo several rounds of cell division, increasing the number of cells (Figure 5 and Video Figure 1).
Retroviral-mediated transfection of a few progenitor cells facilitates the identification of cell clones (F...

Watch this Scientific Journal Video about Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System at JoVE.com

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

라이브 영상 실시간에 세포 행동을 공부 하는 강력한 도구입니다. 여기, 우리는 시간 경과 비디오-현미경 기본 대뇌 피 질 세포의 차별화 된 뉴런을 명과 기본 신경 줄기 세포에서 혈통 진행 동안 제정 단계의 상세한 검사를 허용 하는 프로토콜을 설명 합니다.

2D 문화 시스템을 사용 하 여 기본 대뇌 피 질 세포의 영상 라이브

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

live-imaging-primary-cerebral-cortex-cells-using-2d-culture

Research

JoVE Journal

Neuroscience

8.1K 조회수.  Federal University of Rio Grande do Norte. 라이브 영상 실시간에 세포 행동을 공부 하는 강력한 도구입니다. 여기, 우리는 시간 경과 비디오-현미경 기본 대뇌 피 질 세포의 차별화 된 뉴런을 명과 기본 신경 줄기 세포에서 혈통 진행 동안 제정 단계의 상세한 검사를 허용 하는 프로토콜을 설명 합니다.

비디오: Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

차 쥐 두피 뉴런과 Glia의 Bilaminar의 공동 문화

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

연구

신경과학

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

개발 Neuroepithelium의 세포 문제의 고해상도 라이브 영상

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

기본 교양 Hippocampal 신경에 덴스 코어 Vesicles의 라이브 영상

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

생물학

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

뇌의 Organotypic 슬라이스와 암 세포의 3 차원 회전 타원체와 공 배양 시스템

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

의학

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotypic 슬라이스는 문화 희소 돌기 아교 세포 역학 및 수초화을 공부하기

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

2D 및 3D 인간 유도 만능 줄기 세포 기반 모델은 신코르티컬 개발 중 1 차용성 개입을 해부합니다.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

발생학

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

영상 모니터 세포 생물학과 여러 신경 인구의 계보 진행을 추적 하는 단일 셀 다음 라이브

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

분기 Thalamocortical 축 삭과 시 냅 스의 시각화 Organotypic Cocultures에서 형성

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

통합 된 라이브 셀 모니터링을 통해 인간 유도 만능 줄기 세포 유래 피질 및 도파민 뉴런의 자동화 된 생산

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

 Live Imaging and Single-Cell Tracking to Monitor Neural Lineage in Adult Neural Stem Cells

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/56291/live-imaging-followed-by-single-cell-tracking-to-monitor-cell-biology-and-the-lineage-progression-of-multiple-neural-populations'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>G&#243;mez-Villafuertes, R.,&#160;et. al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>., Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2017)</a> &#160;The video demonstrates the process of preparing, imaging, and tracking adult neural stem cells in real time to study cellular behaviors, including division and differentiation.

Source:&#160;G&#243;mez-Villafuertes, R.,&#160;et. al., Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression ...