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Determining the Influence of Small Molecules on Hypoxic Prostate Cancer Cell (DU-145) Viability Using Automated Cell Counting and a Cell Harvesting Protocol
使用自动细胞计数和细胞收获方案测定小分子对缺氧前列腺癌细胞(DU-145)生存力的影响   

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Abstract

Cell viability assays are an essential aspect of most cancer studies, however they usually require a considerable labor and time input. Here, instead of using the conventional microscopy and hemocytometer cell counting approach, we developed a cell harvesting protocol and combined it with the automated Countess Automated Cell Counter to generate cell viability data. We investigated the effects of dihydroxylated bile acids on the cell viability of prostate cancer cells grown under hypoxic conditions. We observed that for all conditions, cell viability was relatively unchanged, suggesting these molecules had little or no impact on cell viability. The combination of the automated approach and the cell harvesting protocol means this assay is i) easy to perform, ii) extremely reproducible and iii) it complements more conventional cancer assay data such as invasion, migration and adhesion.

Background

Determining the therapeutic utility of any biological molecule is a critical step in the development of novel molecular therapeutics to combat cancer progression and development. As a preliminary step in in vitro characterization, molecules must be assessed for their suitability as anti-cancer therapeutics. As part of this assessment, cell viability is a critical determinant of the cellular response to small molecules as it reflects the ability of a molecule to sustain a threshold cell viability, whilst simultaneously targeting key cancer progression mechanisms e.g., clonogenicity, invasion and adhesion. Conventional viability assays require an intensive labor input comprising microscopy, hemocytometers and manual cell counters. Here we developed a protocol for the rapid and accurate generation of cell viability data that will complement cancer research studies (Phelan et al., 2016).

Materials and Reagents

  1. 15 ml tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  2. Blue 1 ml pipet tips (Greiner Bio One, catalog number: 686295 )
  3. Yellow 200 µl pipet tips (Greiner Bio One, catalog number: 739290 )
  4. Sterile Eppendorf tubes (Eppendorf, catalog number: 0030119401 )
  5. Disposable chamber slide
  6. DU-145 prostate cancer cells (ATCC, catalog number: HTB-81 )
  7. Dulbecco’s modified Eagle’s medium (DMEM) low glucose media (Sigma-Aldrich, catalog number: D5921-500ML )
  8. Deoxycholic acid (Sigma-Aldrich, catalog number: D2510-10G )
  9. 250 mM dimethyloxalylglycine (DMOG) in sterile PBS (EMD Millipore, Calbiochem, catalog number: 400091-50MG )
  10. Chenodeoxycholic acid (Sigma-Aldrich, catalog number: C9377-5G )
  11. Phosphate buffered saline (PBS) tablets (Sigma-Aldrich, catalog number: P4417-100TAB )
  12. Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  13. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F6178-500ML )
  14. Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333-20ML )
  15. Glutamine (Sigma-Aldrich, catalog number: G7513-20ML )
  16. PBS solution (see Recipes)
  17. Complete Dulbecco’s media (see Recipes)
  18. 200 µM DMOG (see Recipes)
  19. CDCA and DCA bile acids (see Recipes)

Equipment

  1. T25 flasks (SARSTEDT, catalog number: 83.3910 )
  2. Cell scrapers (SARSTEDT, catalog number: 83.1831 )
  3. DM0412 centrifuge to accommodate 15 ml tubes (Scilogex, model: DM0412 )
  4. Water jacketed CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: Series II )
  5. Automated pipette filler (Gilson, catalog number: F110753 )
  6. Countess automated cell counter (Thermo Fisher Scientific, InvitrogenTM, catalog number: C10227 )
  7. Inverted microscope (OLYMPUS, model: CKX31 )
  8. Disposable cell chamber slides (Thermo Fisher Scientific, InvitrogenTM, catalog number: C10228 )
  9. Hemocytometer (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10350141 )

Software

  1. Excel

Procedure

  1. Grow DU-145 prostate cancer cells under hypoxic conditions using 200 μM DMOG; DMOG upregulates normoxic HIF-1α by inhibiting HIF-1 prolyl and asparaginyl hydroxylase function at the oxygen dependent degradation domain (ODDD) of the protein. Seed cells at a density of 50,000 cells into T25 flasks in complete Dulbecco’s media and allow them to become confluent over 1-2 days prior to experimentation. Once confluent, remove the media and treat cells with media containing 100 μM bile acids + 200 μM DMOG, DMOG only and media only (untreated controls). Generate enough flasks to ensure viability assessment every 24 h over a 5 day period.
  2. After 24 h, on day 1 of the assay, decant the media, wash the flask surface in sterile PBS to remove debris and gently scrape cells from the flask surface using a cell scraper. Avoid trypsin-EDTA due to potential cytotoxic effects. Add 2 ml of sterile PBS to the flask to create a cell suspension. Mix thoroughly (by gently pipetting up and down) to homogenize cell clumps.
  3. Transfer this 2 ml volume to a 15 ml Falcon tube and centrifuge at 160 x g for 2 min to pellet cells.
  4. Do not fully decant the supernatant, leave approximately 100 µl at the bottom of the tube. Mix this volume gently with the pellet, using a 1 ml blue pipet tip to yield an approximate cell suspension of 100-200 µl. 
  5. Add 20 μl of the cell suspension and 20 μl of trypan blue into a new Eppendorf tube and pipette mix to completely homogenize clumps. Flick mix for a few seconds in order to stain cells.
  6. Load two 10 μl aliquots onto each side of a disposable chamber slide (reusable) and place into the CountessTM automated cell counter to enumerate live/dead cells (viability), represented as percentage cell viability.


    Figure 1. Dihydroxylated bile acids CDCA and DCA do not affect the cell viability of hypoxic DU-145 cells. For cell viability (trypan blue) assays, all cells were grown under hypoxic conditions (DMOG 200 μM), except for untreated cells. No significant compromises were observed in cell viability with respect to time. All data were derived from three independent experiments with at least three replicates in each experiment. Standard deviation was used to generate error bars.

  7. Repeat the procedure each day until data for all 5 days have been collected.

Percentage viability data can be read directly from the automated cell counter. Graph data as viability (%) versus time (day) using Excel (Figure1).

Notes

Percentage viability data can be read directly from the automated cell counter. Graph data as viability (%) versus time (day) using Excel (Figure1).

Data analysis

  1. Trypsin-EDTA should not be used in this protocol as it may induce some cell death which may affect viability thus distorting the data.
  2. Only the DU-145 prostate cancer cell line was tested using this method.
  3. Disposable cell chamber slides can be used 4-5 times (with washing in water) however with time, they become less efficient (bubbles develop in the counting area, distorting counting).
  4. While this assay was carried out in DU-145 prostate cancer cells grown under hypoxic conditions, this assay could be adapted, with some trouble-shooting to those cancer cells grown under normoxic conditions. Indeed, for this assay, untreated DU-145 cells were grown under normoxic conditions and yielded similar viabilities to those of hypoxic test cells (DMOG alone), suggesting hypoxia had little or no impact on viability. The assay could be applicable to any adherent cell line, grown under normoxia or hypoxia conditions, where cell viability is the main measurement output.
  5. To corroborate cell counter data, a hemocytometer was used to manually count cells from random flasks over the duration of the assay (5 days). Overall, an average cell viability of 93.56% ± 2% was recorded suggesting relative parity between methods.
  6. As long as cells are adherent, they can be grown on any sized plate/dish/flask. However, technical issues involving cell scraping may arise on low surface areas.
  7. DU-145 prostate cancer cells are adherent spindle shaped epithelial cells that require passaging every 2-3 days. Typically, cells can be routinely passaged up to passage 20.

Recipes

  1. PBS solution
    Dissolve 5 PBS tablets in 1 L distilled water and autoclave
  2. Complete Dulbecco’s media
    10% fetal bovine serum
    0.5% penicillin-streptomycin
    2 mM glutamine
  3. 200 µM DMOG
    Add 1.15 ml sterile PBS to desiccated powder in bottle
    Mix thoroughly and use at a final concentration of 200 µM
  4. CDCA and DCA bile acids
    50 mM stock solution in distilled water
    Final concentration used: 100 µM

Acknowledgments

This research was supported in part by grants awarded by the European Commission, Science Foundation Ireland, the Department of Agriculture and Food, Ireland, the Irish Research Council for Science, Engineering and Technology and the Health Research Board.

References

  1. Phelan, J. P., Reen, F. J., Dunphy, N., O'Connor, R. and O'Gara, F. (2016). Bile acids destabilise HIF-1α and promote anti-tumour phenotypes in cancer cell models. BMC Cancer 16: 476.

简介

细胞活力测定是大多数癌症研究的一个重要方面,然而,它们通常需要相当多的劳动和时间投入。在这里,而不是使用常规的显微镜和血细胞计数细胞计数方法,我们开发了一个细胞收获协议,并结合自动Countess自动细胞计数器生成细胞活力数据。我们调查二羟基化胆汁酸对缺氧条件下生长的前列腺癌细胞的细胞活力的影响。我们观察到,对于所有条件,细胞活力相对不变,表明这些分子对细胞活力几乎没有或没有影响。自动化方法和细胞收获方案的组合意味着该测定i)容易实施,ii)极其可重复,和iii)其补充更常规的癌症测定数据例如入侵,迁移和粘附。

[背景] 确定任何生物分子的治疗效用是开发新的分子治疗以抵抗癌症进展和发展的关键步骤。作为体外表征的初步步骤,必须评估分子作为抗癌治疗剂的适合性。作为该评估的一部分,细胞活力是小分子的细胞反应的关键决定因素,因为其反映分子维持阈值细胞活力的能力,同时靶向关键癌症进展机制例如。 ,集落性,侵袭和粘附。常规的生存力测定需要包括显微镜,血细胞计数器和手动细胞计数器的大量劳动输入。在这里,我们开发了快速和准确生成细胞活力数据的协议,将补充癌症研究研究(Phelan等人,2016)。

材料和试剂

  1. 15ml管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339650)
  2. 蓝色1ml移液管吸头(Greiner Bio One,目录号:686295)
  3. 黄色200μl移液管吸头(Greiner Bio One,目录号:739290)
  4. 无菌Eppendorf管(Eppendorf,目录号:0030119401)
  5. 一次性腔室滑道
  6. DU-145前列腺癌细胞(ATCC,目录号:HTB-81)
  7. Dulbecco改良的Eagle培养基(DMEM)低葡萄糖培养基(Sigma-Aldrich,目录号:D5921-500ML)
  8. 脱氧胆酸(Sigma-Aldrich,目录号:D2510-10G)
  9. 在无菌PBS(EMD Millipore,Calbiochem,目录号:400091-50MG)中的250mM二甲基草氨酸(DMOG)
  10. 去氧胆酸(Sigma-Aldrich,目录号:C9377-5G)
  11. 磷酸盐缓冲盐水(PBS)片剂(Sigma-Aldrich,目录号:P4417-100TAB)
  12. 台盼蓝(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
  13. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F6178-500ML)
  14. 青霉素 - 链霉素(Sigma-Aldrich,目录号:P4333-20ML)
  15. 谷氨酰胺(Sigma-Aldrich,目录号:G7513-20ML)
  16. PBS溶液(见配方)
  17. 完成Dulbecco的媒体(见配方)
  18. 200μMDMOG(参见配方)
  19. CDCA和DCA胆汁酸(参见配方)

设备

  1. T25烧瓶(SARSTEDT,目录号:83.3910)
  2. 细胞刮刀(SARSTEDT,目录号:83.1831)
  3. DM0412离心机以容纳15ml管(Scilogex,型号:DM0412)
  4. 水夹套CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:系列II)
  5. 自动吸管填充器(Gilson,目录号:F110753)
  6. Countess自动细胞计数器(Thermo Fisher Scientific,Invitrogen TM ,目录号:C10227)
  7. 倒置显微镜(OLYMPUS,型号:CKX31)
  8. 一次性细胞腔玻片(Thermo Fisher Scientific,Invitrogen TM,目录号:C10228)
  9. 血细胞计数器(Thermo Fisher Scientific,Fisher Scientific,目录号:10350141)

软件

  1. Excel

程序

  1. 在缺氧条件下使用200μMDMOG生长DU-145前列腺癌细胞; DMOG通过在蛋白质的氧依赖性降解结构域(ODDD)处抑制HIF-1脯氨酰和天冬酰胺羟基磷酸化功能来上调正常氧HIF-1α。将种子细胞以50,000个细胞的密度接种到完全Dulbecco's培养基中的T25烧瓶中,并使其在实验前1-2天变得汇合。一旦汇合,除去培养基并用含有100μM胆汁酸+200μMDMOG,仅DMOG和仅培养基(未处理的对照)的培养基处理细胞。生成足够的烧瓶,以确保每24小时在5天期间的活力评估。
  2. 24小时后,在测定的第1天,倾析培养基,在无菌PBS中洗涤烧瓶表面以除去碎片,并使用细胞刮刀从烧瓶表面轻轻地刮擦细胞。避免胰蛋白酶-EDTA由于潜在的细胞毒性作用。向烧瓶中加入2ml无菌PBS以产生细胞悬浮液。充分混合(通过轻轻吹打上下),使细胞团匀浆。
  3. 将该2ml体积转移到15ml Falcon管中,并在160×g离心2分钟以沉淀细胞。
  4. 不要完全滗析上清液,留在管底部大约100微升。使用1 ml蓝色移液管头轻轻地将此体积与沉淀混合,得到约100-200μl的细胞悬浮液。
  5. 去除20微升,加入20微升台盼蓝到Eppendorf管和移液管混合物完全匀浆块。轻轻混合几秒钟,以染色细胞
  6. 加载两个10微升等分试样到一次性的腔幻灯片(可重复使用)的每一侧,并放入Countess自动化细胞计数器中枚举活/死细胞(生存力),表示为百分比细胞活力。


    图1.二羟基化胆汁酸CDCA和DCA不影响缺氧DU-145细胞的细胞活力。对于细胞活力(台盼蓝)测定,所有细胞在缺氧条件(DMOG200μM)下生长, ,除了未处理的细胞。在相对于时间的细胞活力中没有观察到显着的折衷。所有数据来自三次独立实验,每次实验至少三次重复。使用标准偏差来产生误差条。

  7. 每天重复该过程,直到收集了所有5天的数据。

百分比活力数据可以从自动细胞计数器直接读取。使用Excel(图1)将图数据作为活力(%)对时间(天)。

笔记

百分比活力数据可以从自动细胞计数器直接读取。使用Excel(图1)将图数据作为活力(%)对时间(天)。

数据分析

  1. 胰蛋白酶-EDTA不应该用于这个协议,因为它可能会诱导一些细胞死亡,可能会影响生存力,从而扭曲数据。
  2. 仅使用该方法测试DU-145前列腺癌细胞系
  3. 一次性细胞腔玻片可以使用4-5次(在水中洗涤),然而随着时间的推移,它们变得效率较低(在计数区域中产生气泡,变形计数)。
  4. 虽然该测定在缺氧条件下生长的DU-145前列腺癌细胞中进行,但是该测定可以改变,对在正常氧条件下生长的那些癌细胞有一些麻烦。实际上,对于该测定,未处理的DU-145细胞在含氧量正常的条件下生长,并产生与缺氧测试细胞(仅DMOG)的那些相似的活力,表明缺氧对生存力具有很小或没有影响。该测定可适用于在含氧量正常或缺氧条件下生长的任何贴壁细胞系,其中细胞活力是主要的测量输出。
  5. 为了证实细胞计数器数据,在测定的持续时间(5天)期间,使用血细胞计数器手动计数来自随机烧瓶的细胞。总体来说,记录93.56%±2%的平均细胞存活率,表明方法之间的相对同等性。
  6. 只要细胞是粘附的,它们可以在任何大小的板/培养皿/烧瓶上生长。然而,涉及细胞刮擦的技术问题可能出现在低表面积上
  7. DU-145前列腺癌细胞是粘附纺锤形上皮细胞,其需要每2-3天传代。通常,细胞可以常规传代至第20代。

食谱

  1. PBS溶液
    将5个PBS片溶于1L蒸馏水和高压釜中
  2. 完成Dulbecco的媒体
    10%胎牛血清 0.5%青霉素 - 链霉素 2mM谷氨酰胺
  3. 200μMDMOG
    加入1.15毫升无菌PBS的干燥的粉末在瓶子
    充分混合并使用最终浓度为200μM的
  4. CDCA和DCA胆汁酸
    50 mM储备液的蒸馏水溶液
    使用的最终浓度:100μM

致谢

这项研究得到了欧盟委员会,爱尔兰科学基金会,爱尔兰农业和食品部,爱尔兰科学,工程和技术研究委员会以及健康研究委员会的赠款的部分支持。

参考文献

  1. Phelan,JP,Reen,FJ,Dunphy,N.,O'Connor,R.and O'Gara,F。(2016)。  胆汁酸使HIF-1α不稳定并促进癌细胞模型中的抗肿瘤表型。 BMC Cancer 16: 476。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Phelan, J. P., Reen, F. and O’Gara, F. (2016). Determining the Influence of Small Molecules on Hypoxic Prostate Cancer Cell (DU-145) Viability Using Automated Cell Counting and a Cell Harvesting Protocol. Bio-protocol 6(22): e2017. DOI: 10.21769/BioProtoc.2017.
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