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XTT Assay of Antifungal Activity
XTT实验检测抗真菌活性   

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Abstract

XTT assay is a colorimetric method that uses the tetrazolium dye, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) to quantify cell-mediated damage to fungi. Actively respiring fungal cells convert the water-soluble XTT to a water-soluble, orange colored formazan product (Meshulam et al., 1995). Here, we describe the protocol that measures the ability of plasmacytoid dendritic cells (pDCs) to exert antifungal activity. This approach was first established with human polymorphonuclear cells (PMN) by Meshulam et al. (1995) and then adapted to pDC by Ramirez-Ortiz et al. (2011) and Loures et al. (2015). It can be modified for use with other effector cells and to test compounds for antifungal activity.

Keywords: Aspergillus fumigatus(烟曲霉), XTT assay(XTT测定), antifungal activity(抗真菌活性), Plasmacytoid dendritic cell(浆细胞样树突细胞)

Material and Reagents

  1. Aspergillus fumigatus (A. fumigatus) clinical isolate Af293 (Nierman et al., 2005)
  2. Human pDCs obtained from healthy donors
  3. RPMI-1640 medium without phenol red (Life Technologies, Gibco®, catalog number: 11875119 )
  4. Penicillin G/ streptomycin sulfate (Life Technologies, Gibco®, catalog number: 15140122 )
  5. L-glutamine (Life Technologies, Gibco®, catalog number: 25030081 )
  6. Sodium pyruvate (Life Technologies, Gibco®, catalog number: 11360070 )
  7. Sterile distilled water (Life Technologies, Gibco®, catalog number: 10977015 )
  8. Sterile PBS (Corning Incorporated, catalog number: 21040CM )
  9. 96 well half area plates (Corning, Costar®, catalog number: 07200309 )
  10. Tips
  11. 15 ml Centrifuge tube (Falcon®,catalog number: 352059 )
  12. XTT (Sigma-Aldrich, catalog number: X4251 )
  13. Coenzyme Q (Sigma-Aldrich, catalog number: D9150 )
  14. Tween 20 (Thermo Fisher Scientific, catalog number: BP337500 )
  15. Sabouraud dextrose agar (Thermo Fisher Scientific, catalog number: R454472 )
  16. Supplemented media (see Recipes)
  17. Slants (see Recipes)

Equipment

  1. 30 μm Nylon mesh (Genesee Scientific Corporation, catalog number: 57105 )
  2. 10 ml Syringe (BD Bioscience, catalog number: 305482 )
  3. Inverted microscope (Nikon)
  4. Pipettes
  5. Multichannel pipette
  6. Neubauer chamber
  7. Reagents reservoir (Corning, Costar®, catalog number: 07200127 )
  8. Microplate reader with capacity to measure OD450 and OD650 (Versamax)
  9. Water bath (Thermo Fisher Scientific)
  10. CO2 Incubator (37 °C)
  11. Vortex (Fisher Vortex Genie 2) (Scientisfic Industries, Inc., catalog number: 12812 )
  12. Autoclavec

Procedure

  1. Grow A. fumigatus (Af293) on Sabouraud dextrose agar slants for 3-5 days at 37 °C. The culture will turn white as hyphae grow and then green as sporulation (conidia formation) occurs.
    Note: The assay can be adapted for other species of fungi.
  2. Harvest the conidia with 2-3 ml of PBS containing 0.05% Tween 20 (PBS T20) gently removing the conidia from the slants with a tip. The conidia can be kept at 4 °C for up to 7 days before use in assays. Maintain sterile conditions when working with conidia.
  3. Suspend the conidia by mixing on a vortex for few seconds. Draw the suspension into a 10 cc syringe. Fold 30 μm nylon mesh inside the top of a 15 ml centrifuge tube. Filter the conidial suspension drop wise through the nylon mesh. Remove and discard the nylon mesh from the centrifuge tube.
  4. Wash the conidia three times by suspension in 10 ml PBS T20 followed by centrifugation at 3,000 rpm/10 min.
  5. Suspend the filtered conidia in 5 ml supplemented media.
  6. Count the conidia in a Neubauer chamber.
  7. Add 5 x 103 A. fumigatus conidia in 96-well half area plates in 100 μl supplemented media per well. If desired for a dose response curve, the number of conidia per well can be varied; however, fewer conidia may not give adequate OD readings.
  8. Keep the plates in the dark at room temperature (21 °C) in static conditions for 16 h in supplemented media to swell the conidia and then incubate for an additional 3 h at 37 °C to promote germination of hyphae. Note that after putting the plates in the incubator check the germination process every 15 min until you get hyphae of 10-20 μm average length.
  9. Withdraw the plate from the incubator when you get hyphae of 10-20 μm average length. If your pDCs are not ready yet keep the plate at 4 °C to avoid overgrowing of the hyphae. We suggest 2 h as the maximum period of storage at 4 °C.
  10. Gently wash the hyphae 2 times with sterile PBS (200 µl).
  11. Add 5 x 104 pDCs to the hyphae in a final volume of 100 μl supplemented media. Note that you can use a different number of pDCs or another effector cell type. Generally, each condition is tested in at least triplicate. Keep the co-culture under sterile conditions.
  12. To determine background activity, 4 to 5 wells should contain pDC only.
  13. To determine XTT reduction by fungi in the absence of pDC, 4 to 5 wells should be left with A. fumigatus hyphae only.
  14. To determine blank values, 4 to 5 wells should have media only.
  15.  Following 2 h incubation at 37 °C, the pDCs are subjected to hypotonic lysis by three washes with 100 μl distilled water. It is critical that the washes are performed very gently and carefully to avoid removing hyphae.
  16. Incubate for 30 min with 100 μl distilled water at 37 °C.
  17. During the 30 min of incubation, the RPMI-1640 containing 400 μg/ml of XTT and 50 μg/ml of Coenzyme Q must be prepared. Stock solutions each containing 10 mg/ml XTT and Coenzyme Q should be prepared individually in PBS. Both compounds need high temperature to dissolve. Usually a 1 min incubation in a 60 °C water bath is sufficient. When more than 1 min is necessary, it is important to take the solutions out of the water bath (at 60 °C) immediately after they dissolve. The XTT and Coenzyme should be added to the RPMI-1640 a few minutes before adding to the wells. As phenol red can interfere with colorimetric readings, the RPMI-1640 should not contain phenol red.
  18. Remove the supernatants, again taking great care taken not to remove the hyphae.
  19. 100 μl RPMI media containing 400 μg/ml of XTT and 50 μg/ml of Coenzyme Q, are added, and the wells are incubated for 2 h at 37 °C.
  20. The OD450 and OD650 are measured on the microplate reader.
  21. Data are expressed as percent antifungal activity according to the formula:

    ODAf + pDC is (OD450 - OD650) of wells containing A. fumigatus hyphae with pDCs.
    ODpDC is (OD450 - OD650) of wells containing pDCs.
    ODAf is (OD450 - OD650) of wells containing A. fumigatus hyphae alone.
    ODblank is (OD450 - OD650) of wells containing media alone.

Recipes

  1. Supplemented media
    RPMI-1640 (without phenol red) with 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate
  2. Slants
    Add 4.5 of Sabouraud dextrose agar to 100 ml distillated water
    Autoclave it for 15 min
    Add 3 ml per 15 ml tube
    Tilt the tube at a 45° to 60° angle and let it solidify. Keep at 4 °C until use.

Acknowledgments

This work was supported in part by National Institutes of Health Grants RO1HL112671, RO1AI025780, and RO1AI072195. FVL acknowledges support from Fundação de Amparo Pesquisa de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

References

  1. Loures, F. V., Rohm, M., Lee, C. K., Santos, E., Wang, J. P., Specht, C. A., Calich, V. L., Urban, C. F. and Levitz, S. M. (2015). Recognition of Aspergillus fumigatus hyphae by human plasmacytoid dendritic cells is mediated by dectin-2 and results in formation of extracellular traps. PLoS Pathog 11(2): e1004643.
  2. Meshulam, T., Levitz, S. M., Christin, L. and Diamond, R. D. (1995). A simplified new assay for assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanil ide (XTT). J Infect Dis 172(4): 1153-1156.
  3. Nierman, W. C., Pain, A., Anderson, M. J., Wortman, J. R., Kim, H. S., Arroyo, J., Berriman, M., Abe, K., Archer, D. B., Bermejo, C., Bennett, J., Bowyer, P., Chen, D., Collins, M., Coulsen, R., Davies, R., Dyer, P. S., Farman, M., Fedorova, N., Fedorova, N., Feldblyum, T. V., Fischer, R., Fosker, N., Fraser, A., Garcia, J. L., Garcia, M. J., Goble, A., Goldman, G. H., Gomi, K., Griffith-Jones, S., Gwilliam, R., Haas, B., Haas, H., Harris, D., Horiuchi, H., Huang, J., Humphray, S., Jimenez, J., Keller, N., Khouri, H., Kitamoto, K., Kobayashi, T., Konzack, S., Kulkarni, R., Kumagai, T., Lafon, A., Latge, J. P., Li, W., Lord, A., Lu, C., Majoros, W. H., May, G. S., Miller, B. L., Mohamoud, Y., Molina, M., Monod, M., Mouyna, I., Mulligan, S., Murphy, L., O'Neil, S., Paulsen, I., Penalva, M. A., Pertea, M., Price, C., Pritchard, B. L., Quail, M. A., Rabbinowitsch, E., Rawlins, N., Rajandream, M. A., Reichard, U., Renauld, H., Robson, G. D., Rodriguez de Cordoba, S., Rodriguez-Pena, J. M., Ronning, C. M., Rutter, S., Salzberg, S. L., Sanchez, M., Sanchez-Ferrero, J. C., Saunders, D., Seeger, K., Squares, R., Squares, S., Takeuchi, M., Tekaia, F., Turner, G., Vazquez de Aldana, C. R., Weidman, J., White, O., Woodward, J., Yu, J. H., Fraser, C., Galagan, J. E., Asai, K., Machida, M., Hall, N., Barrell, B. and Denning, D. W. (2005). Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438(7071): 1151-1156.
  4. Ramirez-Ortiz, Z. G., Lee, C. K., Wang, J. P., Boon, L., Specht, C. A. and Levitz, S. M. (2011). A nonredundant role for plasmacytoid dendritic cells in host defense against the human fungal pathogen Aspergillus fumigatus. Cell Host Microbe 9(5): 415-424.

简介

XTT测定是使用四唑鎓染料,2,3-双 - (2-甲氧基-4-硝基-5-磺酰基) - (2H) - 四唑鎓-5-甲酰苯胺(XTT)定量细胞介导的损伤的比色法 到真菌。 主动呼吸真菌细胞将水溶性XTT转化为水溶性橙色甲an产物(Meshulam等人,1995)。 在这里,我们描述测量浆细胞样树突状细胞(pDCs)发挥抗真菌活性的能力的协议。 该方法首先由Meshulam等人(1995)的人多形核细胞(PMN)建立,然后由Ramirez-Ortiz等人(2011)修改为pDC, Loures 等人(2015)。 其可以被修饰以与其他效应细胞一起使用并且测试化合物的抗真菌活性。

关键字:烟曲霉, XTT测定, 抗真菌活性, 浆细胞样树突细胞

材料和试剂

  1. (烟曲霉)临床分离物Af293(Nierman等人,2005)
  2. 从健康捐献者获得的人pDCs
  3. 无酚红的RPMI-1640培养基(Life Technologies,Gibco ,目录号:11875119)
  4. 青霉素G /链霉素硫酸盐(Life Technologies,Gibco ,目录号:15140122)
  5. L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030081)
  6. 丙酮酸钠(Life Technologies,Gibco ,目录号:11360070)
  7. 无菌蒸馏水(Life Technologies,Gibco ,目录号:10977015)
  8. 无菌PBS(Corning Incorporated,目录号:21040CM)
  9. 96孔半面板(Corning,Costar ,目录号:07200309)
  10. 提示
  11. 15ml离心管(Falcon ,目录号:352059)
  12. XTT(Sigma-Aldrich,目录号:X4251)
  13. 辅酶Q(Sigma-Aldrich,目录号:D9150)
  14. 吐温20(Thermo Fisher Scientific,目录号:BP337500)
  15. Sabouraud右旋糖琼脂(Thermo Fisher Scientific,目录号:R454472)
  16. 补充介质(见配方)
  17. 斜面(见配方)

设备

  1. 30μm尼龙网(Genesee Scientific Corporation,目录号:57105)
  2. 10ml注射器(BD Bioscience,目录号:305482)
  3. 倒置显微镜(尼康)
  4. 移液器
  5. 多通道移液器
  6. 腔室
  7. 试剂库(Corning,Costar ®,目录号:07200127)
  8. 具有测量OD 450和OD 650的能力的微孔板读数器(Versamax)
  9. 水浴(Thermo Fisher Scientific)
  10. CO 2 培养箱(37℃)
  11. 涡旋(Fisher Vortex Genie 2)(Scientisfic Industries,Inc.,目录号:12812)
  12. 高压灭菌器

程序

  1. 成长A。 烟曲霉(Af293)在Sabouraud葡萄糖琼脂斜面上在37℃下培养3-5天。 培养物将变成白色作为菌丝生长,然后绿色作为孢子形成(分生孢子形成)发生。
    注意:该测定可适用于其他真菌种类。
  2. 用2-3ml含0.05%Tween 20的PBS(PBS T20)收获分生孢子,用尖端轻轻地从斜面上除去分生孢子。 在用于测定之前,分生孢子可以在4℃保持长达7天。 当使用分生孢子时,保持无菌条件。
  3. 通过在涡旋上混合几秒钟悬浮分生孢子。 将悬浮液吸入10cc注射器。 在15ml离心管顶部折叠30μm尼龙网。 通过尼龙网过滤分生孢子悬浮液。 从离心管中取出并丢弃尼龙网。
  4. 通过悬浮在10ml PBS T20中然后以3,000rpm/10min离心来洗涤分生孢子三次
  5. 将过滤的分生孢子悬浮在5ml补充的培养基中
  6. 在Neubauer室中计数分生孢子。
  7. 在96孔半面积平板中每孔加入100μl补充的培养基中的5×10 3个烟曲霉分生孢子。如果需要剂量反应曲线,每孔的分生孢子数目可以改变;然而,较少的分生孢子可能不能提供足够的OD读数
  8. 保持板在黑暗中室温(21°C)在静态条件下16小时在补充培养基中膨胀分生孢子,然后在37°C孵育另外3小时,以促进菌丝的发芽。注意,将平板放入孵化器后,每15分钟检查一次发芽过程,直到得到平均长度为10-20微米的菌丝。
  9. 当您获得平均长度为10-20微米的菌丝时,从培养箱中取出平板。如果你的pDC没有准备好,保持板在4℃,以避免菌丝的过度生长。我们建议2小时作为在4℃下的最大储存期
  10. 用无菌PBS(200μl)轻轻洗涤菌丝2次。
  11. 在终体积为100μl的补充培养基中,将5×10 4 pDC加入菌丝中。 请注意,您可以使用不同数量的pDCs或其他效应细胞类型。 通常,每个条件至少三次测试。 保持共培养在无菌条件下。
  12. 为了确定背景活性,4至5个孔应仅含有pDC。
  13. 为了在不存在pDC的情况下确定真菌的XTT减少,4至5个孔应留下 A。 烟曲霉菌丝
  14. 为了确定空白值,4到5个孔应该只有培养基。
  15. 在37℃下孵育2小时后,通过用100μl蒸馏水洗涤三次来对pDC进行低渗裂解。 非常轻柔和小心地进行洗涤以避免移除是至关重要的 菌丝。
  16. 在37℃下用100μl蒸馏水孵育30分钟
  17. 在孵育30分钟期间,必须制备含有400μg/ml XTT和50μg/ml辅酶Q的RPMI-1640。每个含有10mg/ml XTT和辅酶Q的储备溶液应在PBS中单独制备。两种化合物需要高温溶解。通常在60℃水浴中孵育1分钟就足够了。当需要超过1分钟时,重要的是溶解后立即将溶液从水浴(60℃)中取出。在添加到孔中之前,应该将XTT和辅酶加入RPMI-1640几分钟。由于酚红会干扰比色读数,所以RPMI-1640不应含有酚红
  18. 取出上清液,再次小心不要取出菌丝。
  19. 加入100μl含有400μg/ml XTT和50μg/ml辅酶Q的RPMI培养基,并将孔在37℃下孵育2小时。
  20. 在酶标仪上测量OD 450和OD 650。
  21. 数据表示为抗真菌活性百分比,按照公式:

    OD sub + pDC sub是含有A的孔的OD OD 450 OD OD 650。 烟曲霉菌丝与pDC OD pDC 是含有pDC的孔的(OD 450 OD OD 650)。
    OD sub是含有A的孔的(OD 450 OD OD 650)。 烟曲霉单丝。
    OD空白是仅含有培养基的孔的OD OD(OD 450 -OD 650)。< br />

食谱

  1. 补充媒体
    用100U/ml青霉素,100U/ml链霉素,2mM L-谷氨酰胺和1mM丙酮酸钠的RPMI-1640(无酚红)
  2. 斜线
    将4.5沙氏葡萄糖琼脂加入到100ml蒸馏水中 高压灭菌15分钟
    每15 ml管加入3 ml
    将管以45°至60°的角度倾斜并让其固化。 保持在4°C直到使用。

致谢

这项工作部分由国立卫生研究院拨款RO1HL112671,RO1AI025780和RO1AI072195支持。 FVL承认Fundaçãode Amparo Pesquisa deSãoPaulo(FAPESP)和Conselho Nacional de DesenvolvimentoCientíficoeTecnológico(CNPq)的支持。

参考文献

  1. Loures,F.V.,Rohm,M.,Lee,C.K.,Santos,E.,Wang,J.P.,Specht,C.A.,Calich,V.L.,Urban,C.F.and Levitz,S.M。 承认 由人类浆细胞样树突状细胞产生的烟曲霉菌丝由dectin-2介导并导致细胞外陷阱的形成。 PLoS Pathog 11(2):e1004643。
  2. Meshulam,T.,Levitz,S.M.,Christin,L.and Diamond,R.D。(1995)。 用四唑鎓染料评估真菌细胞损伤的简化新测定法,(2,3) - (2-甲氧基-4-硝基-5-磺酰基) - (2H) - 四唑-5-甲酰胺(XTT)。 Infect Dis 172(4):1153 -1156。
  3. Nierman,WC,Pain,A.,Anderson,MJ,Wortman,JR,Kim,HS,Arroyo,J.,Berriman,M.,Abe,K.,Archer,DB,Bermejo,C.,Bennett, Bowieer,P.,Chen,D.,Collins,M.,Coulsen,R.,Davies,R.,Dyer,PS,Farman,M.,Fedorova,N.,Fedorova,N.,Feldblyum,TV,Fischer, R.,Fosker,N.,Fraser,A.,Garcia,JL,Garcia,MJ,Goble,A.,Goldman,GH,Gomi,K.,Griffith-Jones,S.,Gwilliam,R.,Haas,B 。,Haas,H.,Harris,D.,Horiuchi,H.,Huang,J.,Humphray,S.,Jimenez,J.,Keller,N.,Khouri,H.,Kitamoto,K.,Kobayashi,T Konzack,S.,Kulkarni,R.,Kumagai,T.,Lafon,A.,Latge,JP,Li,W.,Lord,A.,Lu,C.,Majoros,WH,May, ,BL,Mohamoud,Y.,Molina,M.,Monod,M.,Mouyna,I.,Mulligan,S.,Murphy,L.,O'Neil,S.,Paulsen,I.,Penalva,MA,Pertea ,M.,Price,C.,Pritchard,BL,Quail,MA,Rabbinowitsch,E.,Rawlins,N.,Rajandream,MA,Reichard,U.,Renauld,H.,Robson,GD,Rodriguez de Cordoba,S 。,Rodriguez-Pena,JM,Ronning,CM,Rutter,S.,Salzberg,SL,Sanchez,M.,Sanchez-Ferrero,JC,Saunders,D.,Seeger,K.,Squares, 。,Takeuchi,M.,Tekaia,F.,Turner,G.,Vazquez de Aldana,CR,Weidman,J.,White,O.,Woodward,J.,Yu,JH,Fraser,C.,Galagan,JE ,Asai,K.,Machida,M.,Hall,N.,Barrell,B.and Denning,DW(2005)。 致病性和致敏丝状真菌的基因组序列烟曲霉 a> 438(7071):1151-1156。
  4. Ramirez-Ortiz,Z.G.,Lee,C.K.,Wang,J.P.,Boon,L.,Specht,C.A.and Levitz,S.M。 浆细胞样树突状细胞在抗人类真菌病原体的宿主防御中的非冗余作用烟曲霉(Aspergillus fumigatus)/em>。 Cell Host Microbe 9(5):415-424。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Loures, F. V. and Levitz, S. M. (2015). XTT Assay of Antifungal Activity. Bio-protocol 5(15): e1543. DOI: 10.21769/BioProtoc.1543.
  2. Loures, F. V., Rohm, M., Lee, C. K., Santos, E., Wang, J. P., Specht, C. A., Calich, V. L., Urban, C. F. and Levitz, S. M. (2015). Recognition of Aspergillus fumigatus hyphae by human plasmacytoid dendritic cells is mediated by dectin-2 and results in formation of extracellular traps. PLoS Pathog 11(2): e1004643.
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