XTT实验检测抗真菌活性

Flávio V. Loures; Stuart M. Levitz

doi:10.21769/BioProtoc.1543

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Peer-reviewed

XTT Assay of Antifungal Activity

XTT实验检测抗真菌活性

Flávio V. Loures email

Stuart M. Levitz

发布: 2015年08月05日第5卷第15期 DOI: 10.21769/BioProtoc.1543 浏览次数: 10469

评审: Emilia Krypotou Belen SanzFanglian He

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Cover of PLOS Pathogens, featuring study using the protocol.

Feb 2015

实验方案合集

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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)

Materials and Reagents

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

Equipment

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