使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Biology
 

Summary

此过程说明如何使用基因脉冲MXcell电系统,以迅速和容易地识别小鼠胚胎成纤维细胞(MEFs)或其他主要细胞的最佳电条件。故障排除注意事项还讨论了相关的视频。

Cite this Article

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McCoy, A. M., Collins, M. L., Ugozzoli, L. A. Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency. J. Vis. Exp. (35), e1662, doi:10.3791/1662 (2010).

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Abstract

这是越来越明显,电是最有效的方法,引入的原代细胞的质粒DNA或siRNA。基因脉冲MXcell电系统和基因脉冲电击缓冲液(BIO - RAD)专门开发的,很容易转染哺乳动物细胞和难以转染的细胞,如小学和干细胞,的核酸。我们将演示如何执行一个简单的实验,以迅速确定最佳电条件。我们将演示如何通过电条件的范围,这样一个实验可以在同一时间进行优化执行运行中的几个样本。我们还将展示最佳条件,确定如何使用96孔电板可以使用标准电试管,促进电板开关,电试管,同时保持相同的电效率。在录像中,我们还将讨论一些可能导致电击实验的成功或失败的的关键因素。

Protocol

1)细胞制备

  1. 当使用贴壁细胞,它是必要trypsinize和收集细胞前电的。
  2. 为了比较各转染小鼠胚胎成纤维细胞(MEF)的文化,代表三种不同的通道号码,,在每个烧瓶中执行以下。
  3. 吸脱落细胞培养基。
  4. 添加PBS洗细胞。
  5. 删除PBS,补充足够的胰蛋白酶盖细胞,并等待几分钟,使胰蛋白酶来分离细胞。
  6. 用显微镜检查验证条件下的细胞培养瓶,嫌烧瓶中分离细胞,然后再次检查烧瓶,确保细胞都分离。
  7. 等待更多的时间和必要时重复。
  8. 所有的细胞中分离后,加入含药血清的介质中和胰蛋白酶。
  9. 细胞转移到离心管中,离心沉淀细胞(RCF = 300 XG)。
  10. 删除已知体积的PBS上清和悬浮细胞。
  11. 计数细胞。
  12. 转移到一个新的管适当体积的细胞悬液,以提供所需数量的细胞进行实验(您将需要在1 × 10 6细胞/ ml密度细胞每孔150μL)。
  13. 离心机细胞。
  14. 取出上清和悬浮细胞在基因脉冲电缓冲区实现了1 × 10 6细胞/ ml的细胞密度适当的音量。
  15. 加入质粒的细胞悬液,每毫升20微克,轻轻混匀。

2)电穿孔容器的设置和电

板的设置和电

  1. 插入基因的脉冲MXcell电系统的电源模块板腔。
  2. 移液器150μL细胞的混合物或缓冲到一个96孔的电板井。
  3. 板腔和脉冲沽盘。
  4. 从室取出钢板。
  5. 吹打在每口井的向上和向下混合以及内容。
  6. 从每口井预热的缓冲液在12孔板的细胞转移。
  7. 塔盘分发细胞和它放在孵化器。
  8. 让细胞恢复为24小时。

试管设置和电

  1. 拔下电源模块板的基因脉冲MXcell电系统和插件在ShockPod™比色皿室室。
  2. 吸取600μL的细胞悬液到0.4厘米的空隙电比色皿。
  3. 比色皿在ShockPod室,并提供电脉冲。
  4. 从室中取出比色皿。
  5. 吹打在比色皿的向上和向下混合比色皿的内容。
  6. 从每口井预热的缓冲液在12孔板的细胞转移。
  7. 塔盘分发细胞和它放在孵化器。
  8. 让细胞恢复为24小时。

3)代表性的成果

在转染细胞,并允许他们重新后,定性分析转染效率,使用啶显微镜,并定量,用流式细胞仪检测。

图1
图1。已成功电穿孔和现在的绿色荧光蛋白基因表达的细胞出现啶显微镜下。

图2
图2。查看相衬下的细胞,使转染和未转染细胞的可视化。这些细胞暴露在200V的最低电压电脉冲。细胞主要是由于高密度融合。

图3
图3。epifluorescence下的同一领域的细胞表达绿色荧光蛋白标记的数量,但这些都只是一个很小的比例在前面的图像中可见的细胞。

图4
图4。250V,相衬下看到活细胞总数略有下降。

图5
图5。epifluorescence,人们可以看到,绿色荧光蛋白表达细胞的数量增加。

图6
图6的最高电压应用,375V,有明显的活细胞较少。

图7
图7。然而,剩下的细胞的很大比例是表达GFP 。取决于哪些条件是最佳的实验设计。在一些实验中的转染的细胞数量最多的可能是最优的,在其他实验的比例最高转可能是最好的。

我们是在细胞,GFP阳性每个条件下,如何的百分比与细胞年龄的不同而有所差异的百分比感兴趣。流式细胞仪可以提供有关每一个不同的电击条件下转染结果的定量信息。

图8
图8:这里的细胞,绿色荧光蛋白在5 12电条件下的每个细胞都表明通过积极的百分比。最大的转染百分率约80%的测试电压最高的指数衰减脉冲,条件6,和70%下最强的方波脉冲下,条件12。

图9
图9。转百分比的总体格局与细胞通过电穿孔前的9倍,几乎是相同的的,但在转染的百分比略有下降。

图10
图10这里显示的是在通过显示一个相对年轻的细胞在转染的百分比显着下降的13个细胞,这些细胞的GFP细胞的百分比。转百分比最高约一半证明后尽快隔离尽可能使用健康细胞的重要性与年轻细胞来实现的。

用于转染MEF的使用基因脉冲MXcell细胞的电穿孔条件
条件(1-6)
指数衰减脉冲,
所有350用友,1000ohm
电压(V)
1 200
2 250
3 300
4 326
5 350
6 376
条件(7-12)
方波脉冲,
所有与2000年用友,1000欧姆,和1个脉冲
电压(V) 脉冲持续时间(毫秒)
7 200 10
8 250 10
9 300 10
10 200 20
11 250 20
12 300 20

Discussion

此视频文章演示了如何使用MXcell电系统可以轻松地找到MEFs或其他主要细胞株的最佳电条件。 96孔板格式允许对许多重复实验或优化的条件下同时进行,这可以消除许多不同的实验需要。在进行此过程中,人们应该记住使用健康细胞后尽快隔离,尽可能使用电条件,匹配电缓冲区。

Disclosures

作者采用Bio - Rad公司实验室产生本文中使用的试剂和仪器

Materials

Name Company Catalog Number Comments
Gene Pulser® Electroporation Buffer Bio-Rad 165-2676
Gene Pulser MXcell™ Electroporation System Bio-Rad 165-2670
Gene Pulser MXcell™ ShockPod™ Cuvette Chamber Bio-Rad 165-2673
Gene Pulser MXcell™ Electroporation System With ShockPod™ Cuvette Chamber Bio-Rad 165-2674

DOWNLOAD MATERIALS LIST

Comments

9 Comments

  1. We are using a Gene Pulser II to transfect MEFs. Do you have a recommendation for conditions to try?
    Thank you,

    Reply
    Posted by: Anonymous
    July 15, 2011 - 10:34 AM
  2. MEFs and other primary cells can optimize at different conditions due to variation among cells and differences in the methods used to generate the MEFs. For best results, we recommend optimizing to identify the best conditions for your experiment. There is a good chance that you will achieve success with the conditions used here although they may not be the absolute best conditions for your particular MEFs. I would recommend using 375V and 350 uF on your GP II with 600 ul of cells resuspended in the Gene Pulser electroporation buffer in a 0.4 cm cuvette as a good starting point. Alternatively, ²50V and 500 uF capacitance would be another good starting point. With a gene pulser II you will want to set the voltage to either 375V or ²50V then set the capacitance to high capacitance setting. The high capacitance settings need to be multiplied by 1000 to get units of uF, so a setting of 0.35 is 350 uf and 0.5 for 500 uF. I hope that helps!

    Reply
    Posted by: Anonymous
    July 19, 2011 - 1:56 PM
  3. We are using a gene pulser (Bio-Rad, Richmond, CA) to transfect adipose tissue stem cells. We want to transfect the cells by electroporation (²50 V, 500 v, 900 v and 1500 v, pulse time 30 ms), but we dont know which capacitance, current and resistane is approperiatefor this. Do you have any recommendation in this regard?
    Thank you

    Reply
    Posted by: Anonymous
    September 3, 2011 - 6:41 AM
  4. Hello, Thank you for your inquiry. It seems you are interested in using a square wave protocol. Bio-Rad Gene Pulser systems can use either exponential or square wave protocols. The set up will be slightly different depending on which system you have (Gene Pulser I, Gene Pulser II, Gene Pulser Xcell or Gene Pulser MXcell) and also the buffer you will be using. We would suggest you try optimizing conditions using the wide range of voltages you have listed. Generally speaking, we have found lower voltages and higher capacitance settings to be best for difficult to transfect cells (such as stem cells). A starting point for a square wave protocol might be ²50V, 950 uF, 30 ms pulse. You can similarly try a wide range of conditions using exponential decay pulses. A starting point for exponential protocols might be ²50V, ²00uF, and 1000 ohms.

    Reply
    Posted by: Anonymous
    March 14, 2012 - 11:32 AM
  5. We are using the Gene Pulser Xcell system. Must we use the Gene Pulser electroporation buffer to get a high transfection efficiency? And where can I find the optimal capicitance for the medium or buffer I am currently using? Moreover, how will the size of the cuvettes and volume of cell suspension influence the transfection efficiency?

    Reply
    Posted by: Anonymous
    February 20, 2012 - 3:00 AM
  6. Crystal, thank you for the questions. It depends on the type of cells you are working with. Gene Pulser electroporation buffer is recommended for mammalian cells especially difficult to transfect cells including primary and stem cells. Other buffers may be sufficient as well. Gene Pulser electroporation buffer can yield both high transfection efficiency and high viability in difficult cell lines. Because of the electrical properties of the buffer it also gives pretty consistent results which helps greatly with optimization.

    Both size of the cuvette and volume of cell suspension should also been taken into consideration when optimizing electroporation conditions as these will alter the electric fields. You can achieve essentially the same electric field through different combinations of parameters. The goal is to find the conditions which work best with your cells and your biological question. If you change the volume of cell suspension you will need to change the conditions applied to achieve the same electrical field. I normally recommend determining the volume and cuvette size you need first, and then optimizing the instrument settings.

    Reply
    Posted by: Anonymous
    March 14, 2012 - 11:33 AM
  7. I am setting up an electroporation experiment using mouse primary MEF and my question is...using the Gene pulser MXcell, I want to know if one condition is good for a 0.4 cuvette size, would the same condition be good for 0.² cuvette size for the electroporation expt.

    Reply
    Posted by: saiphone w.
    August 16, 2012 - 2:48 PM
  8. Hi,
    Thanks for the video. I actually have a problem with getting good viability for urine iPSC. We've been trying to do a successful electroporation with has indicated episomal plasmids using AmaxaTm basic nucleofectorTM kit for primary mammalian epithelial cells, program we used is T-020 from LONZA) but it seems that the GFP vector isn't doing its job very well as we're having trouble to have our Urine iPSC to grow after electroporated using the gene pulser from bio rad)...
    anyways have any suggestions or ideas as to why is this happening?

    Thank you!

    Reply
    Posted by: Emily R.
    January 15, 2014 - 4:19 AM
  9. Hi,
    we want to use Amaxa Nucleofector to transfect CMT93 cells (mouse colorectal cancer). Do you have a recommendation for conditions to try?
    Thank you!

    Reply
    Posted by: Vasiliki Z.
    April 10, 2016 - 9:59 AM

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