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Aug 2018

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Evaluation of Anticancer activity of Silver Nanoparticles on the A549 Human Lung Carcinoma Cell Lines through Alamar Blue Assay
通过阿尔玛蓝显色法评估A549人肺癌细胞系上的银纳米粒子   

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Abstract

Silver nanoparticles have been widely studied to possess antimicrobial as well as anticancer activity, and have found its applications in various fields including pharmaceutical industry, diagnostics, drug delivery, food industry, and others. For this purpose, several cell proliferation assays are widely used for the evaluation of anticancer activity of synthetic compounds as well as natural plant extracts. In general, a compound is said to possess an anticancer activity if it prevents the cancer cells to grow and divide actively, and indirectly activates the generic program of cell death. In this protocol, Alamar blue and MTT assay are described for the analysis of metabolic function and health of the cell. These procedures are generally used for the endpoint analysis. A549 cells are seeded in a 96-well plate, and after the adherence of the cells, they are treated with different concentrations of silver nanoparticles. Followed by 24 h of incubation, colorimetric dyes are added to the wells, and the absorbance is recorded to quantify the percentage cytotoxicity in the sample wells.

Keywords: Silver nanoparticles (银纳米粒子), Cell viability (细胞活力), Alamar blue (阿尔玛蓝), MTT (MTT), Anticancer activity (抗肿瘤活性), Cell proliferation (细胞增殖), Redox indicator (氧化还原指示剂)

Background

Alamar blue has been commonly used for cell viability and cytotoxicity studies in various biological and environmental systems. There are several other commercially available tetrazolium salts that are frequently used as an indirect measure for viable cells. Some of them includes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium, 3’-[1-phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitrobenzene) sulfonic acid hydrate (XTT), and water-soluble tetrazolium salt (WST-1). Besides, Alamar blue and MTT are considered to be the most appropriate, reliable and economic methods. These offer certain advantages including ease of use, increased sensitivity and accuracy, rapid indication of cell toxicity. Both are employed on colorimetric detection of the number of viable cells based on the cellular metabolism activity.

Resazurin, an active component in Alamar blue assay monitors the reducing environment of living cell. The dye acts as an electron acceptor in electron transport chain without disrupting the normal transfer of electrons. As soon as the dye accepts the electrons, it gets reduced to pink color, fluorescent resorufin state. The change in the reduced state can be quantitatively measured spectrophotometrically by recording the absorbance at 570 and 600 nm wavelengths (Rampersad, 2012). Fluorescent signals can also be determined at an excitation wavelength of 530-560 nm and emission wavelength of 590 nm. In the current protocol, anticancer activity of silver nanoparticles was evaluated in A549 lung carcinoma cell lines through Alamar blue assay (Figure 1 A; Kumari et al., 2016; Arya et al., 2018).

In MTT assay (Figure 1 B), the tetrazolium salt is reduced to insoluble formazan dye by dehydrogenase enzyme present in the viable cells at 37 °C. Further, the insoluble formazan salt is dissolved by the addition of solubilizing agents, and the colored product is quantitatively measured spectrophotometrically. The dead cells lose the ability to reduce tetrazolium salts and fail to convert into colored formazan products. Thus, the intensity of the colored product is directly proportional to the number of viable cells present in the culture (Präbst et al., 2017).


Figure 1. Schematic representation of general procedure for cytotoxicity assays using either Alamar blue (A) or MTT reagent (B)

Materials and Reagents

  1. Materials
    1. Pipette tips (Thermo Fisher Scientific, catalog numbers: 90030130-P, 90030230-C)
    2. 96-well tissue culture plates (Himedia, catalog number: TPP96 1X100NO)
    3. Tissue Culture Flask with filter caps (TPP, catalog number: 90026)
    4. Sterile 15 ml centrifuge tubes (Abdos, catalog number: P10402)
    5. 0.22 µm filter (Himedia, catalog number: SF9 1X75NO) 

  2. Cells
    A549 Human lung carcinoma cell lines (NCCS)
    Note: These cell lines are cultured in complete medium containing DMEM media with 10% FBS, 1% pen-strep and are maintained at 37 °C, 5% CO2 in an incubator.

  3. Reagents
    1. DMEM medium (Thermo Fisher Scientific, catalog number: 12100046)
    2. Penicillin-Streptomycin (Pen-Strep) (Himedia, catalog number: A007)
    3. Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: 11573397)
    4. 0.25% Trypsin-EDTA (Thermo Fisher Scientific, catalog number: 25200056)
    5. Dulbecco’s Phosphate Buffer Saline (Himedia, catalog number: TL1022)
    6. Resazurin sodium salt (Alamar Blue) (Sigma-Aldrich, catalog number: R7017)
    7. MTT (Himedia, catalog number: TC247 5X30MG)
    8. Dimethylformamide solution (Sisco Research Laboratories, catalog number: 16616)
    9. Glacial acetic acid (Sisco Research Laboratories, catalog number: 90868)
    10. Sodium dodecyl sulfate (Sisco Research Laboratories, catalog number: 32096)
    11. Resazurin (0.15 mg/ml) stock solution in PBS (see Recipes)
      Note: Resazurin is a light-sensitive dye, so should be stored in the dark.
    12. MTT solution (5 mg/ml) in PBS (see Recipes)
      Note: It should be light protected, and stored at -20 °C for long-term storage.
    13. Solubilization buffer (see Recipes)

Equipment

  1. Micropipettes (SL-20,SL-200, SL-1000, Rainin) 
  2. Balance (XB 220A Precisa, UK)
  3. Hemocytometer (Marienfeld, catalog number: 0610010) 
  4. CO2 Incubator (Panasonic, Multigas Incubator) 
  5. Biosafety cabinet level 2 (CHC Lab, CHC-777A2-04 Biolus)
  6. Inverted Microscope (Zeiss, Primovert)
  7. Benchtop centrifuge (Remi)
  8. Elisa Plate Reader (Thermo Scientific, Thermo Multiskan GO)
  9. U.V. Visible Spectrophotometer (Thermo Scientific, model: Evolution 201)

Procedure

  1. Alamar blue assay
    1. Harvest the cells when they are in log phase of growth and count the cells using a hemocytometer when the cells in the flask are confluent (Figure 2).


      Figure 2. Cell counting. A. Load the hemocytometer chamber by pipetting the cell suspension under the coverslip. B. Place the hemocytometer under the microscope and count the cells.

    2. Seed the cells in a 96-well plate at a cell density of 1 x 104 cells per well in 100 µl complete medium, so that 70-80% confluency will be attained after 1 day (Figure 3).


      Figure 3. Cell seeding. A. Dilute and seed the wells to a cell density of 1 x 104 cells. B. The figure represents a complete seeded micro-well plate.

    3. Keep the seeded plate in a CO2 incubator at 37 °C, 5% CO2, 95% humidity for 24 h to facilitate growth and adherence of cells to the plate surface (Figure 4).


      Figure 4. Incubate the 96-well plate in a CO2 incubator

    4. Wash each well twice with 1x PBS and treat the cells with different concentrations of silver nanoparticles in 100 µl complete medium. Include proper controls of blank medium, untreated seeded wells. Incubate the cells for another 24 h in an incubator (Figure 5).


      Figure 5. Treatment of cells with silver nanoparticles

    5. Add Alamar blue aseptically in an amount equal to 10% volume of the wells (Figure 6).


      Figure 6. Add Alamar blue to each well

    6. Incubate it for 2-4 h in a CO2 incubator. The optimum incubation time varies with cell type and seeding density.
    7. Determine the cell toxicity and proliferation by recording the absorbance at wavelength 570 nm and 600 nm after incubation. Complete media without cells, including Alamar blue, should be considered as blank (Figure 7).

      Spectrophotometric determination of cell proliferation through Alamar blue assay
      The difference in percentage reduction of cells between treated and control cells:



      where,
      E1 = Molar extinction coefficient (E) of oxidized Alamar blue at 570 nm, 80586
      E2 = Molar extinction coefficient of oxidized Alamar blue at 600 nm, 117216
      A1 = Absorbance of sample wells at 570 nm
      A2 = Absorbance of sample wells at 600 nm
      B1 = Growth control absorbance at 570 nm
      B2 = Growth control absorbance at 600 nm

      Fluorescence-based measurement of cell toxicity through Alamar blue assay
      Observe the fluorescence at excitation and emission wavelength of 560 and 590 nm respectively.




      Figure 7. Absorbance recorded through Elisa plate reader

  2. MTT assay
    1. Freshly harvest the cells in cell culture flask and count the cells using a hemocytometer when the cells in the flask become confluent.
    2. Seed the cells in a 96-well microplate at a cell density of 1 x 104 with 100 µl complete medium per well.
    3. Incubate the plate at 5% CO2, 37 °C, 95% humidity for 24 h till the culture in the plate is 70%-80% confluent.
    4. Wash each well with 1x PBS and treat the cells with varying concentrations of silver nanoparticles. Take proper controls, including blank and untreated wells. Keep the plate in the incubator for another 24 h.
    5. Add MTT reagent to an amount equal to 10% of the total volume of wells. For a total volume of 100 µl, add 10 µl of MTT reagent to the wells, including the controls.
    6. Incubate the plate for 2-4 h again (Incubation time varies with the type of cell lines used for the study).
    7. After the formation of formazan crystals during incubation, add 100 µl of solubilization buffer to the wells. 
    8. Stir gently to ensure proper solubilization.
    9. Record the absorbance at 570 nm using spectrophotometer or ELISA plate reader. Measure the reference wavelength higher than 650 nm to calculate the background noise of cell debris and undissolved particles.

      Spectrophotometric determination of cell proliferation through MTT assay



  3. Merits and Limitations
    MTT method is an endpoint analysis method, though it is one of the most widely investigated methods, it has limitations owing to its cytotoxic nature. It has been reported that the formed formazan crystals puncture the cell membrane during exocytosis, thus harming the cell. On the contrary, Alamar blue allows certain advantages over MTT assay including time course measurement analysis, increased sensitivity and accuracy, no cell lysis and is suitable for post-measurement functional studies. A basic disadvantage of using tetrazolium and resazurin dye for the evaluation of cytotoxicity is there need to incubate the cells with these reduction dyes to produce the signals. This tends to result in an increased probability of occurrence of artifacts due to the interaction between the test compound, chemical used for the assay and the cell biochemistry.

Data analysis

The % Reduction of Alamar blue is calculated as per the formula described in previous section.



An example is shown in the following:
E1 = 80586
E2 = 117216
A1 = 0.53
A2 = 0.25
B1 = 0.82
B2 = 0.13



This represents the amount of reduction in treated wells is 55.25% as compared with the control wells. In other words, the cell growth has been inhibited by 44.4% by the test compound, indicating its anticancer activity at a particular concentration.

Notes

  1. All the experiments should be done in duplicates to obtain the statistically significant data, i.e., P ≤ 0.05.
  2. Optimize factors such as plating density, incubation time before starting any experiment because incubation time and cell density varies with the type of cell lines used for the study.

Recipes

  1. Resazurin (0.15 mg/ml) stock solution in PBS
    1. Weigh 1.5 mg resazurin in a 15 ml sterile centrifuge tube and add 10 ml sterile PBS to it
    2. Rotate the tube until the resazurin gets completely dissolved
    3. Filter sterilize the stock solution into a new 15 ml sterile centrifuge tube
    4. The stock solution should be kept at 2-8 °C for long-term storage
  2. MTT solution (5 mg/ml) in PBS
    1. Dissolve 5 mg MTT in 1 ml of Dulbecco’s Phosphate Buffer Saline, pH 7.4
    2. Filter sterilize the MTT solution through a 0.22 µm filter into a fresh sterile container
    3. Store the solution at -20 °C for long-term storage and at 4 °C for frequent use
  3. Solubilization buffer
    1. Make 40% (v/v) Dimethylformamide (DMF) solution in 2% (v/v) glacial acetic acid
    2. Add 16% (w/v) Sodium dodecyl sulfate (SDS) to the solution, and mix it properly
    3. Adjust the pH of the whole solution to 4.7
    4. To avoid the precipitation of SDS, store the buffer at room temperature

Acknowledgments

Surendra Nimesh acknowledges the financial assistance from Department of Biotechnology (DBT), (grant No. 6242-P82/RGCB/PMD/DBT/SNMH/2015) Government of India. This protocol has been modified from one of our previous publication in Arya et al. (2018). Artif Cells Nanomed Biotechnol 46(5): 985-993.

Competing interests

The authors declare that they have no conflicting interests.

References

  1. Arya, G., Kumari, R. M., Gupta, N., Kumar, A., Chandra, R. and Nimesh, S. (2018). Green synthesis of silver nanoparticles using Prosopis juliflora bark extract: reaction optimization, antimicrobial and catalytic activities. Artif Cells Nanomed Biotechnol 46(5): 985-993.
  2. Kumari, R. M., Thapa, N., Gupta, N., Kumar, A. and Nimesh, S. (2016). Antibacterial and photocatalytic degradation efficacy of silver nanoparticles biosynthesized using Cordia dichotoma leaf extract. Advances in Natural Science: Nanoscience and Nanotechnology 7(4) 045009.
  3. Präbst, K., Engelhardt, H., Ringgeler, S. and Hubner, H. (2017). Basic colorimetric proliferation assays: MTT, WST, and resazurin. Methods Mol Biol 1601: 1-17.
  4. Rampersad, S. N. (2012). Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Basel) 12(9): 12347-12360.

简介

银纳米粒子已被广泛研究以具有抗微生物和抗癌活性,并已发现其在各种领域中的应用,包括制药工业,诊断学,药物递送,食品工业等。为此目的,几种细胞增殖试验广泛用于评价合成化合物以及天然植物提取物的抗癌活性。一般而言,如果化合物阻止癌细胞生长和活跃分裂,并且间接激活细胞死亡的一般程序,则据说该化合物具有抗癌活性。在该方案中,描述了Alamar蓝和MTT测定用于分析细胞的代谢功能和健康。这些程序通常用于终点分析。将A549细胞接种在96孔板中,并在细胞粘附后,用不同浓度的银纳米颗粒处理它们。随后孵育24小时,将比色染料加入孔中,记录吸光度以定量样品孔中的细胞毒性百分比。
【背景】Alamar蓝通常用于各种生物和环境系统中的细胞活力和细胞毒性研究。有几种其他商业上可获得的四唑盐经常用作活细胞的间接测量。其中一些包括3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT),4- [3-(4-碘苯基)-2-(4-硝基苯基)-2H-5 -tetrazolio] -1,3-苯二磺酸钠,3' - [1-苯基氨基] - 羰基] -3,4-四唑鎓] - 双(4-甲氧基-6-硝基苯)磺酸水合物(XTT)和水 - 可溶的四唑盐(WST-1)。此外,Alamar blue和MTT被认为是最合适,最可靠和最经济的方法。它们具有某些优点,包括易于使用,提高灵敏度和准确度,快速指示细胞毒性。两者都用于基于细胞代谢活性的活细胞数量的比色检测。

刃天青是Alamar蓝试验中的活性成分,可监测活细胞的还原环境。染料在电子传输链中充当电子受体而不破坏电子的正常转移。一旦染料接受电子,它就会变成粉红色,荧光试剂。通过记录570和600nm波长处的吸光度,可以通过分光光度法定量测量还原状态的变化(Rampersad,2012)。荧光信号也可以在530-560nm的激发波长和590nm的发射波长下测定。在目前的方案中,通过Alamar蓝测定在A549肺癌细胞系中评估银纳米颗粒的抗癌活性(图1A; Kumari 等人,2016; Arya 等。,2018)。

在MTT测定中(图1B),四唑盐通过活细胞中存在的脱氢酶在37℃还原成不溶的甲dye染料。此外,通过添加增溶剂溶解不溶性甲salt盐,并通过分光光度法定量测量有色产物。死细胞失去了减少四唑盐的能力,无法转化为有色甲product产品。因此,有色产物的强度与培养物中存在的活细胞数成正比(Präbst et al。,2017)。


图1.使用Alamar蓝(A)或MTT试剂(B)进行细胞毒性测定的一般程序的示意图

关键字:银纳米粒子, 细胞活力, 阿尔玛蓝, MTT, 抗肿瘤活性, 细胞增殖, 氧化还原指示剂

材料和试剂

  1. 材料
    1. 移液器吸头(Thermo Fisher Scientific,目录号:90030130-P,90030230-C)
    2. 96孔组织培养板(Himedia,目录号:TPP96 1X100NO)
    3. 带过滤盖的组织培养瓶(TPP,目录号:90026)
    4. 无菌15毫升离心管(Abdos,目录号:P10402)
    5. 0.22μm过滤器(Himedia,目录号:SF9 1X75NO) 

  2. 细胞
    A549人肺癌细胞系(NCCS)
    注意:这些细胞系在含有10%FBS,1%pen-strep的DMEM培养基的完全培养基中培养,并在培养箱中维持在37℃,5%CO 2 。 / em>

  3. 试剂
    1. DMEM培养基(Thermo Fisher Scientific,目录号:12100046)
    2. 青霉素 - 链霉素(Pen-Strep)(Himedia,目录号:A007)
    3. 胎牛血清(Thermo Fisher Scientific,目录号:11573397)
    4. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,目录号:25200056)
    5. Dulbecco的磷酸盐缓冲盐水(Himedia,目录号:TL1022)
    6. 刃天青钠盐(Alamar Blue)(Sigma-Aldrich,目录号:R7017)
    7. MTT(Himedia,目录号:TC247 5X30MG)
    8. 二甲基甲酰胺溶液(Sisco Research Laboratories,目录号:16616)
    9. 冰醋酸(Sisco Research Laboratories,目录号:90868)
    10. 十二烷基硫酸钠(Sisco Research Laboratories,目录号:32096)
    11. 刃天青(0.15 mg / ml)在PBS中的储备液(参见食谱)
      注意:刃天青是一种光敏染料,因此应存放在黑暗中。
    12. PBS中的MTT溶液(5mg / ml)(参见配方)
      注意:它应该是光保护的,并存储在-20°C下长期存放。
    13. 增溶缓冲液(见食谱)

设备

  1. 微量移液器(SL-20,SL-200,SL-1000,Rainin) 
  2. Balance(XB 220A Precisa,UK)
  3. 血细胞计数器(Marienfeld,目录号:0610010) 
  4. CO 2 孵化器(Panasonic,Multigas Incubator) 
  5. 生物安全柜2级(CHC实验室,CHC-777A2-04 Biolus)
  6. 倒置显微镜(Zeiss,Primovert)
  7. 台式离心机(Remi)
  8. Elisa Plate Reader(Thermo Scientific,Thermo Multiskan GO)
  9. 紫外光可见分光光度计(Thermo Scientific,型号:Evolution 201)

程序

  1. Alamar蓝测定
    1. 当细胞处于生长的对数期时收获细胞,并在烧瓶中的细胞汇合时使用血细胞计数器计数细胞(图2)。


      图2.细胞计数。 :一种。通过移取盖玻片下的细胞悬液加载血细胞计数器室。 B.将血细胞计数器放在显微镜下并计数细胞。

    2. 将细胞在96孔板中以100μl完全培养基中每孔1×10 4个细胞密度接种,使1天后达到70-80%汇合(图3) )。


      图3.细胞接种。 A.将孔稀释并接种至细胞密度为1×10 4个细胞。 B.该图代表完整的种子微孔板。

    3. 将接种的平板保持在37℃,5%CO 2 ,95%湿度的CO 2 培养箱中24小时,以促进细胞生长和细胞粘附到板表面(图4)。


      图4.将96孔板在CO 2 培养箱中孵育

    4. 用1x PBS洗涤每个孔两次,并在100μl完全培养基中用不同浓度的银纳米颗粒处理细胞。包括适当的空白培养基,未处理的种子孔。将细胞在培养箱中孵育24小时(图5)。


      图5.用银纳米粒子处理细胞

    5. 无菌添加Alamar蓝,其量等于孔体积的10%(图6)。


      图6.在每个孔中加入Alamar蓝色

    6. 在CO 2 培养箱中孵育2-4小时。最佳培养时间随细胞类型和接种密度而变化。
    7. 通过记录孵育后在570nm和600nm波长处的吸光度来确定细胞毒性和增殖。没有细胞的完整培养基,包括Alamar蓝,应视为空白(图7)。

      通过Alamar蓝测定分光光度法测定细胞增殖
      处理细胞和对照细胞之间细胞减少百分比的差异:



      在哪里,
      E1 =氧化的Alamar蓝在570nm处的摩尔消光系数(E),80586
      E2 = 600nm处氧化的Alamar蓝的摩尔消光系数,117216
      A1 =样品孔在570nm处的吸光度
      A2 =样品孔在600nm处的吸光度
      B1 = 570nm处的生长控制吸光度
      B2 = 600nm处的生长控制吸光度

      通过Alamar蓝测定法对细胞毒性进行荧光测定
      观察激发和发射波长分别为560和590 nm的荧光。




      图7.通过Elisa读板器记录的吸光度

  2. MTT检测
    1. 在细胞培养瓶中新鲜收获细胞,并在烧瓶中的细胞融合时使用血细胞计数器计数细胞。
    2. 将细胞在细胞密度为1×10 4 /μL的96孔微孔板中培养,每孔100μl完全培养基。
    3. 将平板在5%CO 2 ,37℃,95%湿度下孵育24小时,直至平板中的培养物为70%-80%汇合。
    4. 用1x PBS洗涤每个孔并用不同浓度的银纳米颗粒处理细胞。采取适当的控制措施,包括空白和未经处理的井。将培养皿中的培养板再保持24小时。
    5. 加入MTT试剂,其量等于孔总体积的10%。对于总体积为100μl,向孔中添加10μlMTT试剂,包括对照。
    6. 再次孵育平板2-4小时(孵育时间随用于研究的细胞系类型而变化)。
    7. 在孵育过程中形成甲crystals晶体后,向孔中加入100μl增溶缓冲液。 
    8. 轻轻搅拌以确保适当的溶解。
    9. 使用分光光度计或ELISA板读数器记录570nm处的吸光度。测量高于650 nm的参考波长,以计算细胞碎片和未溶解颗粒的背景噪音。

      通过MTT法分光光度法测定细胞增殖



  3. 优点和局限
    MTT法是一种终点分析方法,虽然它是最广泛研究的方法之一,但由于其细胞毒性,它具有局限性。据报道,形成的甲crystals晶体在胞吐作用期间刺穿细胞膜,从而损害细胞。相反,Alamar blue比MTT测定具有一定的优势,包括时程测量分析,灵敏度和准确度提高,无细胞裂解,适用于测量后功能研究。使用四唑鎓和刃天青染料评估细胞毒性的基本缺点是需要用这些还原染料孵育细胞以产生信号。由于测试化合物,用于测定的化学品和细胞生物化学之间的相互作用,这倾向于导致伪影发生的可能性增加。

数据分析

Alamar蓝的%减少量按照前一节中描述的公式计算。



下面显示了一个示例:
E1 = 80586
E2 = 117216
A1 = 0.53
A2 = 0.25
B1 = 0.82
B2 = 0.13



这表示与对照孔相比,处理孔的减少量为55.25%。换句话说,试验化合物使细胞生长受到44.4%的抑制,表明其在特定浓度下的抗癌活性。

笔记

  1. 所有实验都应重复进行,以获得统计学上显着的数据,即, P ≤0.05。
  2. 在开始任何实验之前优化诸如平板密度,孵育时间等因素,因为孵育时间和细胞密度随着用于研究的细胞系类型而变化。

食谱

  1. 刃天青(0.15mg / ml)在PBS中的储备液
    1. 在15ml无菌离心管中称取1.5mg刃天青,并向其中加入10ml无菌PBS
    2. 旋转管直至刃天青完全溶解
    3. 过滤将储备溶液灭菌到新的15ml无菌离心管中
    4. 储备溶液应保持在2-8°C,以便长期储存
  2. PBS中的MTT溶液(5mg / ml)
    1. 将5 mg MTT溶于1 ml Dulbecco磷酸盐缓冲盐水(pH 7.4)中
    2. 过滤器将MTT溶液通过0.22μm过滤器灭菌到新鲜的无菌容器中
    3. 将溶液储存在-20°C下长期储存,在4°C下储存以备常用
  3. 增溶缓冲液
    1. 在2%(v / v)冰醋酸中制备40%(v / v)二甲基甲酰胺(DMF)溶液
    2. 向溶液中加入16%(w / v)十二烷基硫酸钠(SDS),并适当混合
    3. 将整个溶液的pH调节至4.7
    4. 为避免SDS沉淀,将缓冲液储存在室温下

致谢

Surendra Nimesh承认印度政府提供的生物技术部(DBT)(第6242-P82 / RGCB / PMD / DBT / SNMH / 2015号授权)的财政援助。该协议已经从我们之前在Arya 等人(2018)中的出版物中修改过。 Artif Cells Nanomed Biotechnol 46(5):985-993。

利益争夺

作者声明他们没有利益冲突。

参考

  1. Arya,G.,Kumari,R.M.,Gupta,N.,Kumar,A.,Chandra,R。和Nimesh,S。(2018)。 使用 Prosopis juliflora 树皮提取物绿色合成银纳米粒子:反应优化,抗菌和催化活性。 Artif Cells Nanomed Biotechnol 46(5):985-993。
  2. Kumari,R.M.,Thapa,N.,Gupta,N.,Kumar,A。和Nimesh,S。(2016)。 使用生物合成的银纳米粒子的抗菌和光催化降解功效Cordia dichotoma 叶子提取物。 自然科学进展:纳米科学和纳米技术 7(4)045009。
  3. Präbst,K.,Engelhardt,H.,Ringgeler,S。和Hubner,H。(2017)。 基本比色增殖试验:MTT,WST和刃天青。 方法Mol Biol 1601:1-17。
  4. Rampersad,S。N.(2012)。 Alamar Blue的多次应用,作为细胞活力生物测定中代谢功能和细胞健康的指标。 传感器(巴塞尔) 12(9):12347-12360。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sharma, N., Arya, G., Kumari, R. M., Gupta, N. and Nimesh, S. (2019). Evaluation of Anticancer activity of Silver Nanoparticles on the A549 Human Lung Carcinoma Cell Lines through Alamar Blue Assay. Bio-protocol 9(1): e3131. DOI: 10.21769/BioProtoc.3131.
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