Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation
利用磺酰罗丹明B（SRB）法检测细胞的增殖
发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.1984 浏览次数: 49515
评审: HongLok LungChunjing QuAnonymous reviewer(s)
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The authors used this protocol in:
Jul 2015
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Abstract
The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be extracted using basic conditions; thus, the amount of bound dye can be used as a proxy for cell mass, which can then be extrapolated to measure cell proliferation.
The protocol can be divided into four main steps: preparation of treatment, incubation of cells with treatment of choice, cell fixation and SRB staining, and absorbance measurement. This assay is limited to manual or semiautomatic screening, and can be used in an efficient and sensitive manner to test chemotherapeutic drugs or small molecules in adherent cells. It also has applications in evaluating the effects of gene expression modulation (knockdown, gene expression upregulation), as well as to study the effects of miRNA replacement on cell proliferation (Kasinski et al., 2015).
Background
The SRB assay has been widely used to investigate cytotoxicity in cell based studies and it is the method of choice for high cost-effective screenings (Vichai and Kirtikara, 2006). Since this method does not rely on measuring metabolic activity [e.g., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT], the steps required to optimize the protocol for a specific cell line are substantially simplified.
The protocol described here has been optimized for medium throughput screening of miRNAs with tumor suppressive properties in adherent lung cancer cells in 96-well format and 384-well format (Kasinski et al., 2015). Particularly the SRB assay in 384-well format offers the advantage of screening a large number of miRNA mimics or compounds in a single plate (> 60 per plate, 6 replicates) using inexpensive equipment and reagents.
Materials and Reagents
- 96-well clear flat-bottom polystyrene tissue-culture plates (Corning, catalog number: 3596 )
- 384-well clear flat-bottom polystyrene tissue-culture plates (Corning, catalog number: 3701 )
- 96-well PCR plates (Corning, Axygen®, catalog number: PCR-96-FS-C )
- 100 mm tissue-culture plates (Corning, catalog number: 430167 )
- 1.5 ml Eppendorf tubes (VWR, catalog number: 89000-028 )
- 15 ml Falcon tubes (Corning, Falcon®, catalog number: 352097 )
- Pipette tips (Mettler-Toledo International, catalog numbers: RT-L10FLR )
- Pipette tips (Mettler-Toledo International, catalog numbers: RT-L200F )
- Pipette tips (Mettler-Toledo International, catalog numbers: RT-L1000F )
- MatrixTM pipette tips (1,250 μl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 8245 )
- MatrixTM pipette tips (125 μl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 7445 )
- Adherent cell line of interest
- Appropriate culture medium
- Reagent reservoir sterile (Corning, Costar®, catalog number: 4870 )
- Reagent reservoir non sterile (VWR, catalog number: 89094-684 )
- Opti-MEM® (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 ) or serum free medium (SFM, appropriate culture medium before adding fetal bovine serum)
- Phosphate buffered saline (PBS) (GE Healthcare, catalog number: SH30256 )
- Trypsin solution (2.5%, wt/vol) (GE Healthcare, catalog number: SH30042.01 )
- Fatal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
- Trypan blue (Sigma-Aldrich, catalog number: T9154 )
- Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: 91228 )
- Sulforhodamine B sodium salt (SRB) (Sigma-Aldrich, catalog number: S1402 ) in 1% (vol/vol) acetic acid
- Acetic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: S25118 )
- 10 mM unbuffered Tris base solution (Sigma-Aldrich)
- DNAse/RNase free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9932 )
- Lipofectamine RNAimax (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13778150 )
- Mirvana miRNA mimics (Thermo Fisher Scientific, AmbionTM)
- miRNA precursor molecules - negative control #2 (non-targeting scramble miRNA) (Thermo Fisher Scientific, AmbionTM, catalog number: AM17111 )
Equipment
- Pipettes (Mettler-Toledo International, catalog numbers: L-1000XLS )
- Pipettes (Mettler-Toledo International, catalog numbers: L200-XLS )
- Pipettes (Mettler-Toledo International, catalog numbers: L20-XLS )
- Pipettes (Mettler-Toledo International, catalog numbers: L2-XLS )
- Multichannel pipettes (Mettler-Toledo International, catalog numbers: L12-200XLS )
- Multichannel pipettes (Mettler-Toledo International, catalog numbers: L12-20XLS )
- MatrixTM multichannel electronic pipette (384-well format) (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 2011 )
- MatrixTM multichannel electronic pipette (96-well format) (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 2004 )
- CO2 incubator (Panasonic Biomedical Sales Europe, model: MCO-230AIC-UV )
- Inverted microscope (Nikon, model: TS100 )
- Multiwell microplate reader (Glomax, Promega)
- Hematocytometer (Hausser Scientific, Bright-LineTM, catalog number: 1492 )
Software
- Statistical analysis software (GraphPad Prism, SPSS)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Orellana, E. A. and Kasinski, A. L. (2016). Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation. Bio-protocol 6(21): e1984. DOI: 10.21769/BioProtoc.1984.
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分类
癌症生物学 > 通用技术 > 细胞生物学试验
细胞生物学 > 细胞活力 > 细胞增殖
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