欢迎您, 登录 | 注册

首页 | English

X
加载中

This protocol utilizes PicoGreen 96-well plate technology. This method is applied to estimate the sensitivity of different tumor cell lines to chemodrugs.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Cell Survival Rate Assay
细胞存活率检测

癌症生物学 > 细胞死亡 > 细胞生物学试验 > 细胞活性
作者: FengZhi Liu
FengZhi LiuAffiliation: School of Biomedical Sciences, Thomas Jefferson University, Philadelphia, USA
For correspondence: fengzhi6@yahoo.com
Bio-protocol author page: a51
Vol 2, Iss 12, 6/20/2012, 7455 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.216

[Abstract] This protocol utilizes PicoGreen 96-well plate technology. This method is applied to estimate the sensitivity of different tumor cell lines to chemodrugs.

Materials and Reagents

  1. 10% FBS culture medium
  2. Chemodrugs
  3. PicoGreen (Life Technologies, Invitrogen™, catalog number: P7581)
  4. TryLE express (Trypsin) (Life Technologies, Gibco®, catalog number: 12605-010)
  5. Phosphate buffered saline (PBS)
  6. Deionized water

Equipment

  1. 96-Well culture plate
  2. TECAN Genios Instrument (PHENIX)
  3. Incubator
  4. Foil
  5. Spectrophotometer

Procedure

  1. Suck out medium, wash once with 5 ml PBS. Add 2 ml tryLE express. After cells become detached, add 10 ml complete medium. Pipette the cell suspension with 5-10 ml pipette to become single cell suspension. This step is very important (to get single cell suspension, put the pipette tip onto the bottom of the flask, then push out the cell suspension through squeezing the cell suspension out of pipette).
  2. Count the cells and dilute to 1 x 10/ml, place cells in 96-well plate in 100 μl (1 x103/well).
  3. Interested medication was diluted in a series of concentrations and was added in wells in 100 μl (the medication was prepared in 2 times of final concentration).
  4. Put the plate in 37 °C, 5% CO2 for 1 week.
  5. Remove media from plate by shaking the plate in sink. Then tap on towel to remove the remaining of media.
  6. Wash with PBS 200 μl/well twice. Remove PBS by shaking plate. Tap plate on towel.
  7. Add 100 μl deionized water to each well. Place the plate in incubator 37 °C, 5% CO2 for 1 h.
  8. Dilute PicoGreen in deionized water 1:200 (will need 100 μl/well). Make sure to calculate how much you will need. Wrap the plate with foil and let sit for 1 h at RT (this step can be done overnight if you are busy).
  9. Measure the plate with spectrophotometer for fluorescence intensity.
  10. Process the data:
    Survival Rate =
    Average fluorescence intensity in experimental wells x 100%
    Average fluorescence intensity in control wells
    Note: PicoGreen is a fluorochrome that selectively binds dsDNA and has characteristics similar to that of SYBR-Green I. It has a maximum excitation at 480 nm and emission at 520 nm.

Acknowledgments

This protocol was adapted from Ahn et al. (1996) and Enger (1996).

References

  1. Ahn, S. J., Costa, J. and Emanuel, J. R. (1996). PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR. Nucleic Acids Res 24(13): 2623-2625.
  2. Enger, O. (1996). Use of the fluorescent dye PicoGreen for quantification of PCR products after agarose gel electrophoresis. Biotechniques 21(3): 372-374.


How to cite this protocol: Liu, F. (2012). Cell Survival Rate Assay. Bio-protocol 2(12): e216. DOI: 10.21769/BioProtoc.216; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册