Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue.
Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue.
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DOI：
10.3791/63007
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发表时间：
2021-10-21
影响因子：
1.2
通讯作者：
Allen, Antino R.
中科院分区：
文献类型：
--
作者：
Trujillo, Madison;McElroy, Taylor;Brown, Taurean;Simmons, Pilar;Ntagwabira, Fabio;Allen, Antino R.
这种神经分离方案(商业成人脑分离试剂盒附带的方案的改编)优化了组织处理，为详细的下游分析做准备，如流式细胞术或单细胞测序。神经解离可以通过机械解离(如使用过滤器、切碎技术或吸管研磨)、酶消化或其组合来进行。神经细胞的微妙性质可能会使获得高度可行的、真正的单细胞悬浮液的努力复杂化，而单细胞分析所需的细胞碎片最少。数据表明，这种自动机械分离和酶消化的组合一致地产生了高度活性(90%)的单细胞悬液，克服了上述困难。虽然有几个步骤需要手动灵活，但这些步骤减少了样品处理和潜在的细胞损失。这份手稿详细说明了这一过程的每一步，以装备其他实验室，成功地分离少量神经组织，为下游分析做准备。这种神经细胞分离方案适用于起始材料含量较低的样品，并产生用于下游分析的高度可行的单细胞悬浮液，并具有可选的固定和染色步骤。
This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrates that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (>90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis. This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.
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影响因子：
3.7
作者：
Stewart, Judith C.;Villasmil, Michelle L.;Frampton, Mark W.
通讯作者：
Frampton, Mark W.
DOI：
10.1007/7651_2014_115
发表时间：
2015-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Rodrigues, Goncalo M C;Fernandes, Tiago G;Diogo, Maria Margarida
通讯作者：
Diogo, Maria Margarida
单细胞分辨率的小鼠大脑 DNA 甲基化图谱。
影响因子：
64.8
作者：
Liu H;Zhou J;Tian W;Luo C;Bartlett A;Aldridge A;Lucero J;Osteen JK;Nery JR;Chen H;Rivkin A;Castanon RG;Clock B;Li YE;Hou X;Poirion OB;Preissl S;Pinto-Duarte A;O'Connor C;Boggeman L;Fitzpatrick C;Nunn M;Mukamel EA;Zhang Z;Callaway EM;Ren B;Dixon JR;Behrens MM;Ecker JR
通讯作者：
Ecker JR
影响因子：
16.2
作者：
Armand EJ;Li J;Xie F;Luo C;Mukamel EA
通讯作者：
Mukamel EA
影响因子：
--
作者：
Li L;Lenahan C;Liao Z;Ke J;Li X;Xue F;Zhang JH
通讯作者：
Zhang JH