The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.
Authors
The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.
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1Department of Biomedical Engineering, University of California, Irvine (UCI), 2Stem Cell Research Center, University of California, Irvine (UCI), 3Institute for Brain Aging and Dementia, University of California, Irvine (UCI)
Harris, J., Lee, H., Tu, C. T., Cribbs, D., Cotman, C., Jeon, N. L. Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device. J. Vis. Exp. (8), e305, doi:10.3791/305 (2007).
在这个视频中，我们展示了E18皮质大鼠神经元的准备。 E18皮质大鼠以前解剖，并准备从E18胎鼠大脑皮层神经元得到。 E18皮层，清扫后，立即到单个神经元的分离。它可以存储长达一个星期的E18皮质休眠发送含有B27的缓冲区4 ° C的分解是执行之前。然而，将细胞活力下降。通常情况下，我们获得我们的E18的Cortex新鲜。在冰冷的无钙镁免费清扫缓冲区（招商基金）被运送到实验室。抵达后，胰蛋白酶添加到皮质的终浓度为0.125％。皮层，然后在37℃孵育8分钟。 DMEM培养液含10％胎牛血清添加到皮质停止反应。然后在2分钟2500转离心皮质。上清被删除，含2％B27（体积/体积）和0.25％Glutamax（体积/体积），然后向上和向下吹打重悬的皮质神经基底媒体2毫升（NBM）。下一步，皮质是磨碎与以前火抛光的玻璃滴管，用较小的一个连续的开放每个。研制过程后，皮层再次离心2分钟2500转。然后取出上清液和皮质颗粒重新悬浮2毫升含有B27与Glutamax的NBM。细胞悬液，然后通过一个40微米的尼龙细胞过滤网。下一步的细胞计数。现正准备装载到神经元的微流体装置的神经元。
准备E18胎鼠皮层神经元为条块
开始之前，重要的是温暖的一切必要的媒体和试剂，以37 ° C
同样重要的是消毒一切准备的细胞（例如，橡胶灯泡，试管架，媒体瓶，等），并用70％乙醇擦拭，这是摆在引擎盖。
注 ：终浓度的细胞通常是250万细胞/毫升和800万细胞/毫升之间
载入细胞
后的细胞已被计算，它是时间的负载设备的细胞：
根据不同的应用，可以是多种多样的细胞密度。然而，播种密度过低的主要神经元通常会导致细胞死亡。根据不同的孵化器和湿度水平，媒体可能需要在设备改为每2至3天。不断变化的媒体时，这是很重要的媒体取出后水库，切勿从媒体的主渠道。此外，新媒体，并放置在顶部的井，让一些新鲜的媒体流通过设备进入主渠道，填补了所有的水塘前，它是很好的做法。
| Name | Type | Company | Catalog Number | Comments |
| NBM | medium | Invitrogen | 21103-049 | Neural Basal Media is obtained by Invitrogen under their Gibco line of cell culture reagents. |
| E18 rat embryos | Animal | Obtained from pregnant Sprague Dawley Rats. The pregnant rat is sacrificed and the E18 fetal rat pups dissected to obtain the E18 fetal rat cortex. | ||
| CMFM dissection buffer HBSS | buffer | ice-cold Calcium-free Magnesium-free dissection buffer HBSS based. | ||
| 15 ml Tube | Tool | BD Biosciences | Falcon No. 352097 | Available through Fisher Scientific catalog number 14-959-70C |
| 0.25% Trypsin-EDTA | Reagent | Invitrogen | ||
| 37˚C water bath | ||||
| Pasteur pipets | Fisher Scientific | glass | ||
| DMEM | medium | Invitrogen | ||
| FBS | Reagent | Invitrogen | serum, used at 10% in DMEM | |
| centrifuge | set at 2500 rpm | |||
| Trypan Blue | Invitrogen | for counting live/dead cells | ||
| BD Falcon Cell Strainer 40 um | Tool | BD Biosciences | Falcon cat# 352340 | Available through Fisher Scientific. Fisher catalog# 08-771-1 |
| B27 | Reagent | Invitrogen | 17504-044 | B27 is a proprietary supplement available through Invitrogen under their Gibco line of cell culture reagents. |
| Glutamax | Reagent | Invitrogen | 35050-061 | Glutamax is available through Invitrogen under their Gibco line of celll culture reagents. |
1. Park, J.W., Vahidi, B., Taylor, A.M., Rhee, S.W., Jeon, N.L. Microfluidic culture platform for neuroscience research. Nat Protoc. 2006;1(4):2128-36.
2. Taylor, A.M., Blurton-Jones, M., Rhee, S.W., Cribbs, D.H., Cotman, C.W., Jeon, N.L. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods. 2005 Aug;2(8):599-605.
This is quite impressive. How do you avoid the neurons in the small channels sticking to the PDMS and peeling off when you remove the slabs before fixation? Is there some sort of special treatment for the PDMS or technique used before removal so the axons are not disturbed?
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ReplyPosted by: AllysonJune 20, 2008, 2:05 PM