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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   

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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

  1. 96-well clear flat-bottom polystyrene tissue-culture plates (Corning, catalog number: 3596 )
  2. 384-well clear flat-bottom polystyrene tissue-culture plates (Corning, catalog number: 3701 )
  3. 96-well PCR plates (Corning, Axygen®, catalog number: PCR-96-FS-C )
  4. 100 mm tissue-culture plates (Corning, catalog number: 430167 )
  5. 1.5 ml Eppendorf tubes (VWR, catalog number: 89000-028 )
  6. 15 ml Falcon tubes (Corning, Falcon®, catalog number: 352097 )
  7. Pipette tips (Mettler-Toledo International, catalog numbers: RT-L10FLR )
  8. Pipette tips (Mettler-Toledo International, catalog numbers: RT-L200F )
  9. Pipette tips (Mettler-Toledo International, catalog numbers: RT-L1000F )
  10. MatrixTM pipette tips (1,250 μl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 8245 )
  11. MatrixTM pipette tips (125 μl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 7445 )
  12. Adherent cell line of interest
  13. Appropriate culture medium
  14. Reagent reservoir sterile (Corning, Costar®, catalog number: 4870 )
  15. Reagent reservoir non sterile (VWR, catalog number: 89094-684 )
  16. Opti-MEM® (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 ) or serum free medium (SFM, appropriate culture medium before adding fetal bovine serum)
  17. Phosphate buffered saline (PBS) (GE Healthcare, catalog number: SH30256 )
  18. Trypsin solution (2.5%, wt/vol) (GE Healthcare, catalog number: SH30042.01 )
  19. Fatal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
  20. Trypan blue (Sigma-Aldrich, catalog number: T9154 )
  21. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: 91228 )
  22. Sulforhodamine B sodium salt (SRB) (Sigma-Aldrich, catalog number: S1402 ) in 1% (vol/vol) acetic acid
  23. Acetic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: S25118 )
  24. 10 mM unbuffered Tris base solution (Sigma-Aldrich)
  25. DNAse/RNase free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9932 )
  26. Lipofectamine RNAimax (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13778150 )
  27. Mirvana miRNA mimics (Thermo Fisher Scientific, AmbionTM)
  28. miRNA precursor molecules - negative control #2 (non-targeting scramble miRNA) (Thermo Fisher Scientific, AmbionTM, catalog number: AM17111 )

Equipment

  1. Pipettes (Mettler-Toledo International, catalog numbers: L-1000XLS )
  2. Pipettes (Mettler-Toledo International, catalog numbers: L200-XLS )
  3. Pipettes (Mettler-Toledo International, catalog numbers: L20-XLS )
  4. Pipettes (Mettler-Toledo International, catalog numbers: L2-XLS )
  5. Multichannel pipettes (Mettler-Toledo International, catalog numbers: L12-200XLS )
  6. Multichannel pipettes (Mettler-Toledo International, catalog numbers: L12-20XLS )
  7. MatrixTM multichannel electronic pipette (384-well format) (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 2011 )
  8. MatrixTM multichannel electronic pipette (96-well format) (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 2004 )
  9. CO2 incubator (Panasonic Biomedical Sales Europe, model: MCO-230AIC-UV )
  10. Inverted microscope (Nikon, model: TS100 )
  11. Multiwell microplate reader (Glomax, Promega)
  12. Hematocytometer (Hausser Scientific, Bright-LineTM, catalog number: 1492 )

Software

  1. Statistical analysis software (GraphPad Prism, SPSS)

Procedure

  1. Treatment solution preparation
    1. Volumes of treatment of choice should be enough for triplicates in 96-well plates (50 μl per replicate; final volume in well 100 μl) or six replicates in 384-well plates (10 μl per replicate; final volume in well 20 μl) and also account for pipetting variation.
    2. Treatment can be prepared in aqueous solution (Opti-MEM for transfections) or solvent of choice (e.g., DMSO).

  2. Cell preparation
    1. Remove medium from cell monolayers and wash the cells once with sterilized PBS.
    2. Remove PBS and add 1 ml (100 mm plates) 0.25% (wt/vol) trypsin to evenly cover the cell-growth surface.
    3. Incubate at 37 °C for 5 min or until cells start to dissociate.
    4. Next inactivate trypsin with 10 volumes of culture medium containing FBS, and mix up and down to obtain a homogeneous single cell suspension.
    5. Transfer the cell suspension to a sterile Falcon tube. Determine the cell concentration by counting in a hematocytometer chamber under a microscope using a 1:1 mixture of cell suspension and 0.4% (wt/vol) trypan blue solution to determine cell viability prior cell seeding. Optional: before counting, spin down cells in order to wash trypsin and resuspend in growth medium.
      Note: Only proceed to next steps if cells in solution look healthy by checking trypan blue staining (Figure1).


      Figure 1. Trypan blue staining of human bronchial epithelial cells (HBEC). The arrow points to a colored cell indicating that the cell is dead. The live cell underneath it, has an intact membrane and thus is not colored.

    6. Adjust the cell concentration with growth medium (10% FBS) to obtain an appropriate cell seeding density per well in a volume of 50 μl (96-well format) or 10 μl (384-well format).
      Note: Initial cell seeding density will depend on two main factors: 1) doubling time of the cell line used and 2) the day the plate will be read. Cells are typically at 70-80% confluency (2 x 104 for 96-well format and 8 x 103 for 384-well format) by the end of the experiment (day 5).
    7. Transfer the cell suspension into a sterile reagent reservoir to make it easier to pipette with a multichannel pipette.

  3. Treatment exposure
    1. Mix the treatment solutions prepared in step A by pipetting. Dispense 50 μl (96-well format) or 10 μl (384-well format) of solution into each well.
    2. Mix cell suspension prepared in step B thoroughly and add 50 μl (96-well format) or 10 μl (384-well format) to each well already containing treatment solutions.
      Note: Ensure even cell distribution in the bottom of the plate and avoid shaking the plate to avoid 'ring effects'. The best way to achieve this is to add the cell solution directly to the bottom of the well, avoiding touching the walls. We also recommend performing a short spin of the plate (20 sec, 10 x g) before placing it in the incubator.
    3. Set aside three wells in the plate containing only Opti-MEM or solvent of choice and cell suspension for an untreated or vehicle control. Also, leave three wells in the plate containing only medium for background subtraction.
    4. Incubate the plate at 37 °C in a humidified incubator with 5% CO2 until plate is to be read.
      Note: The incubation time will depend on the type of compound being tested and has to be experimentally determined. For example, in our hands we observe the biggest difference in cell proliferation between negative control and a tumor suppressive miRNA 5 days following transfection.

  4. Cell fixation and staining
    1. Gently add 25 μl (96-well format) or 5 μl (384-well format) cold 50% (wt/vol) TCA to each well directly to medium supernatant, and incubate the plates at 4 °C for 1 h. Mixing is not required, as this could lead to some cells detaching from the bottom of the well.
      Wash the plates four times by submerging the plate in a tub with slow-running tap water and remove excess water by gently tapping the plate into a paper towel. After the last wash allow the plate to air-dry at room temperature.
      Note: Cell monolayer detachment can occur if water is forced into the wells. We recommend letting the plate dry completely before continuing to next step. If necessary, dried plates can be stored at room temperature indefinitely.
    2. Add 50 μl (96-well format) or 20 μl (384-well format) of 0.04% (wt/vol) SRB solution to each well.
      Note: Ensure that the solution is in direct contact with the bottom of the well and that there are no bubbles in between. SRB solution is not light sensitive thus plate does not need to be covered during incubation.
    3. Leave at room temperature for 1 h and then quickly rinse the plates four times with 1% (vol/vol) acetic acid (200 μl for 96-well format or 30 μl for 384-well format) to remove unbound dye. Allow the plate to air-dry at room temperature.
      Note: Non-homogeneous washes are a major source of error in this assay. Washes must be done quickly and homogeneously across the entire plate. This is particularly important for 384-well format. The small area of the wells leads to high superficial tension sometimes making it difficult to wash the plates uniformly. Visually check that all the wells in the plate have been injected with 1% (vol/vol) acetic acid and that there are no bubbles preventing the washes. We recommend washing with multi-channel pipet and injecting the solution indirectly into the wells using the walls of the wells.

  5. Absorbance measurement
    1. Add 50 μl to 100 μl of 10 mM Tris base solution (pH 10.5) to each well and shake the plate on an orbital shaker for 10 min to solubilize the protein-bound dye (approximately 10 min).
    2. Measure the absorbance at 510 nm in a microplate reader.

Data analysis

Subtract background absorbance from all wells. Calculate the percentage of cell-growth inhibition normalizing treatments to miRNA negative control using the following formula:
% cell growth = Absorbance sample/Absorbance negative control or untreated x 100 (Figure 2B)
% growth inhibition = 100 - % cell growth
SRB assay results can only be used if the data falls within the linearity limit of the assay. We recommend performing a cell number titration experiment to determine the linear dynamic range of the assay with the cell line used (Figure 2A). Generally, O.D. of > 2 are not within the linear range of the assay.
Statistical tests for this type of assay can include t-tests for two group comparisons or ANOVA for multiple group comparisons.



Figure 2. Sulforhodamine B colorimetric assay in cell culture to analyze cell proliferation. A. Cell number titration (H441) using the SRB assay 384-well format, n = 6, error bars = SD. B. miR-34a dose response analysis in lung cancer cell lines H441 and H358 using SRB 96-well format, n = 3.

Notes

  1. In our experience, the use of multichannel electronic pipettes for 384-well format assays reduces variation due to pipetting errors. For that purpose it is highly recommended that miRNA-liposomal complexes to be prepared in 96-well PCR plates, which will allow the use of multichannel pipette.
  2. Seeding density will depend on the doubling time of the cell line to be used as well as the day of the readout. As a rule of thumb, cells should not be allowed to reach 100% confluency by the day of the readout. From our experience, a final confluency of 80% provides satisfactory results with non-small cell lung cancer cell lines (e.g., H441, A549, EKVX).
  3. The day of fixation after treatment must also be determined empirically. In our experience, 4-6 days after transfection of miRNA mimics provides satisfactory results in non-small cell lung cancer cell lines showing maximal growth inhibition compared to controls.
  4. For miRNA transfections:
    1. MiRNA-liposomal complexes can be prepared in serum free/antibiotic free medium.
    2. The amount of Lipofectamine RNAimax must be empirically determined for each cell line to be used in order to identify the amount of reagent that provides the maximum transfection efficiency and minimal cell toxicity. This eliminates the need of changing media after transfection, which is a common source of variation in these types of assays.
    3. In our hands, we determined that 0.12 μl (for 96-well plastes - 100 μl) or 0.05 μl (for 96-well plates - 20 μl) of Lipofectamine RNAimax was enough to transfect 6 nM of miRNA mimic with minimal toxicity in a panel of non-small cell lung cancer cells. Each transfection should be replicated in the same plate (we recommend at least 3 replicates in a 96-well plate or 6 in a 384-well plate).
    4. From our experience, reverse transfection reduces the time of the assay and at the same time reduces sources of variation during changes of media before transfection, washing steps and replacement of Opti-MEM after transfection.

Acknowledgments

The protocol presented herein was adapted from Kasinski et al. (2015). This work was supported by an NIH grant (R00CA178091 and P30CA023168).

References

  1. Kasinski, A. L., Kelnar, K., Stahlhut, C., Orellana, E., Zhao, J., Shimer, E., Dysart, S., Chen, X., Bader, A. G. and Slack, F. J. (2015). A combinatorial microRNA therapeutics approach to suppressing non-small cell lung cancer. Oncogene 34(27): 3547-3555.
  2. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S. and Boyd, M. R. (1990). New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82(13): 1107-1112.
  3. Vichai, V. and Kirtikara, K. (2006). Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1(3): 1112-1116.

简介

Limonium 已知有性和无融合生殖(通过种子的无性繁殖)繁殖模式。在这里,我们提出解剖协议为胚珠的发芽。使用微分干涉对比(DIC)显微镜。该协议允许更好地处理胚珠,并提供优于早期技术,特别是在较大胚珠的某些优势。这种方法还能够观察到完整胚珠中的减数分裂和胚囊发育,以及容易检测到区别性和无序性发育的事件。

[背景] 发生在胚珠发育期间,有必要细胞学检查胚珠。这项研究可以涉及显微镜观察石蜡或树脂包埋,切片材料或清除的器官。在D'Amato(1940; 1949)的先驱作品中公开了对性和无融合生殖物种中胚珠和胚囊发育的第一次细胞学研究。在这些作品中,花使用Karpechenko的方法固定,包埋在石蜡中,切片并用Heidenhain的铁苏木精染色,其染色质和染色体在细胞核中染色。使用这些方法的花芽切片可导致由于单个细胞的部分破坏结构完整性而具有差质量的制备物。更容易的选择是清除福尔马林:乙酸:乙醇固定的器官并用纯的Mayer's hemalum染色(Wallis,1957; Stelly等人,1984)。这种技术需要少得多的时间和劳动,特别是对于通常在卵巢中形成小胚珠的物种,这是 Limonium spp的情况。然而,在小和大胚珠中,水合氯醛作为清除溶液比水杨酸甲酯更好,因为在后者中,液体胚珠变得相当脆弱并且在实验期间难以处理。我们的方法与酶胚消化胚珠有助于揭示中央质量的组织,牛乳腺和其覆盖的两个珠宝,特别是在大胚珠。在微分干涉对比光学下观察到在水合氯醛中清除的减数分裂和精细胚珠和胚囊的实例。...

材料和试剂

  1. 96孔透明平底聚苯乙烯组织培养板(Corning,目录号:3596)
  2. 384孔透明平底聚苯乙烯组织培养板(Corning,目录号:3701)
  3. 96孔PCR板(Corning,Axygen ,目录号:PCR-96-FS-C)
  4. 100mm组织培养板(Corning,目录号:430167)
  5. 1.5ml Eppendorf管(VWR,目录号:89000-028)
  6. 15ml Falcon管(Corning,Falcon ,目录号:352097)
  7. 移液管吸头(Mettler-Toledo International,目录号:RT-L10FLR)
  8. 移液管吸头(Mettler-Toledo International,目录号:RT-L200F)
  9. 移液管吸头(Mettler-Toledo International,目录号:RT-L1000F)
  10. (Thermo Fisher Scientific,Thermo Scientific TM ,目录号:8245)的移液管吸头
  11. Matrix(125μl)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:7445)
  12. 感兴趣的粘附细胞系
  13. 适当的培养基
  14. 试剂库无菌(Corning,Costar ,目录号:4870)
  15. 试剂库无菌(VWR,目录号:89094-684)
  16. Opti-MEM(Thermo Fisher Scientific,目录号:31985070)或无血清培养基(SFM,添加胎牛血清前的适当培养基)
  17. 磷酸盐缓冲盐水(PBS)(GE Healthcare,目录号:SH30256)
  18. 胰蛋白酶溶液(2.5%,wt/vol)(GE Healthcare,目录号:SH30042.01)
  19. 将致死性牛血清(FBS)(Sigma-Aldrich,目录号:F2442)
  20. 台盼蓝(Sigma-Aldrich,目录号:T9154)
  21. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:91228)
  22. 在1%(vol/vol)乙酸中的磺酰罗丹明B钠盐(SRB)(Sigma-Aldrich,目录号:S1402)
  23. 乙酸(Thermo Fisher Scientific,Fisher Scientific,目录号:S25118)
  24. 10mM无缓冲的Tris碱溶液(Sigma-Aldrich)
  25. DNAse/RNAse游离水(Thermo Fisher Scientific,Ambion TM,目录号:AM9932)
  26. Lipofectamine RNAimax(Thermo Fisher Scientific,Invitrogen TM ,目录号:13778150)
  27. Mirvana miRNA mimics(Thermo Fisher Scientific,Ambion TM
  28. miRNA前体分子 - 阴性对照#2(非靶向scramble miRNA)(Thermo Fisher Scientific,Ambion TM ,目录号:AM17111)

设备

  1. 移液器(Mettler-Toledo International,目录号:L-1000XLS)
  2. 移液管(Mettler-Toledo International,目录号:L200-XLS)
  3. 移液管(Mettler-Toledo International,目录号:L20-XLS)
  4. 移液管(Mettler-Toledo International,目录号:L2-XLS)
  5. 多通道移液管(Mettler-Toledo International,目录号:L12-200XLS)
  6. 多通道移液管(Mettler-Toledo International,目录号:L12-20XLS)
  7. Matrix 多通道电子移液管(384孔格式)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2011)
  8. Matrix 多通道电子移液管(96孔格式)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2004)
  9. CO 2 sub孵化器(Panasonic Biomedical Sales Europe,型号:MCO-230AIC-UV)
  10. 倒置显微镜(尼康,型号:TS100)
  11. 多孔微板读数器(Glomax,Promega)
  12. 血细胞计数器(Hausser Scientific,Bright-Line TM ,目录号:1492)

软件

  1. 统计分析软件(GraphPad Prism,SPSS)

程序

  1. 处理溶液制备
    1. 选择的治疗体积应足以在96孔板中一式三份(每个重复50μl;孔100μl中的最终体积)或384孔板中的6个重复(每个重复10μl;孔20μl的终体积)也说明移液变化。
    2. 可以在水溶液(用于转染的Opti-MEM)或所选择的溶剂(例如DMSO)中制备处理。

  2. 细胞制备
    1. 从细胞单层去除培养基,用无菌PBS洗涤细胞一次
    2. 除去PBS,加入1ml(100mm板)0.25%(wt/vol)胰蛋白酶以均匀覆盖细胞生长表面。
    3. 在37℃孵育5分钟或直到细胞开始解离。
    4. 接下来用10体积的含有FBS的培养基使胰蛋白酶失活,并且上下混合以获得均匀的单细胞悬浮液。
    5. 将细胞悬液转移到无菌Falcon管。通过在显微镜下使用细胞悬浮液和0.4%(wt/vol)台盼蓝溶液的1:1混合物在血细胞计数器室中计数来确定细胞浓度,以确定细胞接种前的细胞活力。可选:在计数前,旋转细胞以洗涤胰蛋白酶并重悬于生长培养基中。
      注意:如果通过检查台盼蓝染色(图1),溶液中的细胞看起来健康,则只需进行后续步骤。


      图1.人支气管上皮细胞(HBEC)的台盼蓝染色。箭头指向有色细胞,表明细胞死亡。在它下面的活细胞,具有完整的膜,因此不着色
    6. 用生长培养基(10%FBS)调节细胞浓度,以50μl(96孔板)或10μl(384孔板)的体积每孔获得合适的细胞接种密度。
      注意:初始细胞接种密度将取决于两个主要因素:1)所用细胞系的倍增时间和2)板将被读取的那天。细胞通常在70-80%汇合(对于96-孔形式为2×10 4个,对于384-孔形式为8×10 3个),在实验结束时(第5天)。
    7. 将细胞悬液转移到无菌试剂池,以便更容易用多通道移液器移液
  3. 治疗风险
    1. 通过吸移混合步骤A中制备的处理溶液。在每个孔中加入50μl(96孔板)或10μl(384孔板)溶液
    2. 彻底混合步骤B中制备的细胞悬液,并向已经含有处理溶液的每个孔中加入50μl(96-孔形式)或10μl(384-孔形式)。
      注意:确保平板细胞均匀分布在平板底部,避免摇动平板以避免"环效应"。实现这一点的最佳方法是将细胞溶液直接添加到孔底部,避免接触壁。我们还建议在将板置于培养箱中之前进行短旋转(20秒,10 x g)。
    3. 将仅含有Opti-MEM或选择的溶剂的板中的三个孔,以及用于未处理或载体对照的细胞悬浮液。另外,在仅含有用于背景扣除的培养基的平板中留下三个孔。
    4. 在37℃下在具有5%CO 2的潮湿培养箱中孵育该平板,直到读取平板。
      注意:孵育时间将取决于被测试化合物的类型,并且必须通过实验确定。例如,在我们的手中,在转染后5天,观察到阴性对照和肿瘤抑制性miRNA之间细胞增殖的最大差异。

  4. 细胞固定和染色
    1. 轻轻地添加25微升(96孔格式)或5微升(384孔格式)冷50%(重量/体积)TCA到每个孔直接到培养基上清液,并在4°C孵育板1小时。不需要混合,因为这可能导致一些细胞从孔底部分离 通过将板浸入带有慢速自来水的桶中来洗涤板四次,并通过轻轻地将板轻拍到纸巾中来去除过量的水。最后一次洗涤后,将板在室温下风干 注意:如果水被迫进入孔,则可能发生细胞单层脱离。我们建议让板完全干燥,然后继续下一步。如果需要,干燥的板可以无限期地在室温下储存。
    2. 向每个孔中加入50μl(96-孔形式)或20μl(384-孔形式)的0.04%(wt/vol)SRB溶液。
      注意:确保溶液与孔底部直接接触,并且两者之间没有气泡。 SRB溶液不是光敏感的,因此在孵育期间不需要覆盖板。
    3. 在室温下放置1小时,然后用1%(体积/体积)乙酸(200μl用于96孔板或30μl用于384孔板)快速冲洗板4次以除去未结合的染料。使板在室温下风干。
      注意:非均匀洗涤是该测定中的主要误差来源。必须快速和均匀地在整个板上进行洗涤。这对384孔格式特别重要。孔的小面积导致高的表面张力,有时使得难以均匀地洗涤板。目视检查板中的所有孔已注入1%(vol/vol)乙酸,并且没有气泡防止洗涤。我们建议用多通道移液器洗涤,并使用孔壁将溶液间接注入孔中。

  5. 吸光度测量
    1. 向每个孔中加入50μl至100μl的10mM Tris碱溶液(pH 10.5),并在轨道摇床上摇动板10分钟以溶解结合蛋白的染料(约10分钟)。
    2. 在酶标仪上测量510 nm处的吸光度。

数据分析

减去所有孔的背景吸光度。使用下式计算细胞生长抑制归一化处理对miRNA阴性对照的百分比:
%细胞生长=吸光度样品/吸光度阴性对照或未处理×100(图2B) %生长抑制= 100%细胞生长
SRB测定结果只能在数据落入测定的线性限度内时使用。我们建议进行细胞数滴定实验以确定使用细胞系的测定的线性动态范围(图2A)。一般来说,O.D.的> 2不在测定的线性范围内 这种类型的测定的统计学测试可以包括用于两组比较的试验或用于多组比较的ANOVA。< br /


图2.细胞培养物中的磺酰罗丹明B比色测定以分析细胞增殖。A.使用SRB测定384孔格式的细胞数滴定(H441),n = 6,误差条= SD。 B.使用SRB 96孔形式的肺癌细胞系H441和H358中的miR-34a剂量反应分析,n = 3。

笔记

  1. 根据我们的经验,使用多通道电子移液器384孔格式化验可减少由于移液误差引起的变化。为此,强烈建议在96孔PCR板中制备miRNA-脂质体复合物,这将允许使用多通道移液器。
  2. 接种密度将取决于待使用的细胞系的倍增时间以及读出的天数。作为经验法则,不应允许细胞在读出当天达到100%汇合。根据我们的经验,80%的最终融合提供了非小细胞肺癌细胞系(例如H441,A549,EKVX)的令人满意的结果。
  3. 治疗后的固定日也必须凭经验确定。根据我们的经验,转染miRNA模拟物后4-6天在非小细胞肺癌细胞系中提供令人满意的结果,与对照相比显示最大的生长抑制。
  4. 对于miRNA转染:
    1. 可以在无血清/抗生素的培养基中制备MiRNA-脂质体复合物。
    2. 对于要使用的每种细胞系,必须凭经验确定Lipofectamine RNAimax的量,以鉴定提供最大转染效率和最小细胞毒性的试剂的量。这消除了在转染后改变培养基的需要,这是这些类型的测定的常见变化来源。
    3. 在我们的手中,我们确定在面板中0.12μl(对于96-孔塑料-100μl)或0.05μl(对于96-孔板 - 20μl)的Lipofectamine RNAimax足以转染6nM的miRNA模拟物而毒性最小的非小细胞肺癌细胞。每个转染应在同一板中复制(我们建议在96孔板中至少重复3次,或在384孔板中至少重复3次)。
    4. 根据我们的经验,逆转染减少了测定的时间,同时减少了转染,洗涤步骤和在转染后更换Opti-MEM后培养基更换期间的变异来源。

致谢

本文提供的方案改编自Kasinski等人。 (2015)。这项工作得到了NIH资助(R00CA178091和P30CA023168)的支持。

参考文献

  1. Kasinski,AL,Kelnar,K.,Stahlhut,C.,Orellana,E.,Zhao,J.,Shimer,E.,Dysart,S.,Chen,X.,Bader,AGand Slack,FJ(2015)。   抑制非小细胞肺的组合microRNA治疗方法癌症 癌基因 34(27):3547-3555
  2. Skehan,P.,Storeng,R.,Scudiero,D.,Monks,A.,McMahon,J.,Vistica,D.,Warren,JT,Bokesch,H.,Kenney, 。  抗癌药物筛选的新的比色细胞毒性测定。/a> J Natl Cancer Inst 82(13):1107-1112
  3. Vichai,V.和Kirtikara,K。(2006)。  Sulforhodamine B比色法测定细胞毒性筛选。 Nat Protoc 1(3):1112-1116。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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