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

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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, 7599 views, 0 Q&A
DOI: https://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.

[Abstract] PicoGreen -96孔培养皿的实验步骤。这个方法用来评价不同的肿瘤细胞系对化疗药物的敏感度。

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.

材料与试剂

 

1.        10%的胎牛血清培养基

2.        化疗药物

3.        PicoGreen(一种染料) (Invitrogen, 货号# P7581)

4.        TryLE express(胰岛素), GIBCO, 货号# 12605-010

 

设备

   

1.        1.96孔培养皿

2.        TECAN(帝肯公司) Genios 仪器(PHENIX)

 

实验步骤

 

1.        吸出培养基,用5mlPBS(磷酸盐缓冲液)洗一次,加2mltryLE express,当细胞都分离开来后,加10ml的完全培养基。用5—10ml移液枪悬浮,使之变成单细胞悬浮液。这一步很重要。(为了得到单细胞悬浮液,将移液枪的尖端放到烧瓶的底部吸附,然后慢慢将细胞悬浮液打出以更好的获得单细胞悬浮液)。

2.        计数细胞,然后稀释到1x104/mL96孔培养皿中每孔加100μl稀释好的细胞液(1x103/孔)。

3.        将化疗药物稀释成一系列不同的浓度,然后每个孔中加100μl(因为稀释2倍,所以要按终浓度2倍准备药物浓度)。

4.        将培养皿放置37°C,5% CO2条件下培养一周。

5.        将培养皿在水槽中晃动,以便除去培养基,用滤纸去除残留的培养基。

6.        每个孔用200μlPBS洗涤两次。晃动培养基去除PBS,用滤纸将培养皿上的PBS吸附干净。

7.        在每个孔中加100ul的去离子水,将培养皿放置37°C,5% CO2条件下培养1h

8.        用去离子水按1:200的稀释PicoGreen染料 (每孔需要100ul),提前计算好需要的量。用锡箔纸包裹好培养皿,室温放置1h(如果忙的话,可以放置过夜)。

9.        用分光光度计测量培养皿中每孔的荧光强度。

10.     数据处理:

存活率=  实验组中孔的平均荧光强度/对照组中孔的平均荧光强度x100%

注:PicoGreen 是一种可以选择性的与双链DNA结合的荧光染料,与SYBR-Green I染料性质相似,在480nm处被激发,在520nm处放射光波。

 

参考文献:

 

1.         Ahn S.J., Costa J., Emanuel J.R. (1996). PicoGreen quantitation of DNA: Effective evaluation of samples pre- or post-PCR. Nucleic Acids Res 24(13): 2623-5. 

2.        Enger O. (1996). Use of the fluorescent dye picogreen for quantification of PCR products after agarose gel electrophoresis. Biotechniques 21(3): 372-4. 

 

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



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