Abstract
as medidas tradicionais de atividade antimicrobiana intracelular e citotoxicidade de célula eucariótica contar com ensaios de ponto de extremidade. Tais ensaios de ponto final requerem vários passos experimentais adicionais antes da leitura, tais como lise celular, a determinação da unidade de formação de colónias, ou adição de reagentes. Ao realizar milhares de ensaios, por exemplo, durante a triagem de alto rendimento, a jusante do esforço necessário para estes tipos de ensaios é considerável. Portanto, para facilitar a descoberta de alto rendimento antimicrobianos, foi desenvolvido um ensaio em tempo real simultaneamente para identificar inibidores de crescimento bacteriano intracelular e avaliar a citotoxicidade de células eucarióticas. Especificamente, a detecção de crescimento bacteriano intracelular em tempo real foi habilitado pela marcação estirpes bacterianas de triagem, quer com um operon bacteriano lux (1 ensaio de geração de st) ou repórteres de proteínas fluorescentes (de 2ª geração, ensaio ortogonal). Um não-tóxico, célula de membrana-impermeabilizante, corante de ligação de ácido nucleicoTambém foi adicionado durante a infecção inicial de macrófagos. Estes corantes são excluídos a partir de células viáveis. No entanto, as células hospedeiras não viáveis perder a integridade da membrana permitindo a entrada e marcação fluorescente de DNA nuclear (ácido desoxirribonucléico). Notavelmente, ADN de ligação está associado a um grande aumento no rendimento quântico fluorescente que fornece uma leitura à base de solução de morte da célula hospedeira. Temos utilizado este ensaio combinado para executar uma tela de alto rendimento em formato de microplacas, e para avaliar o crescimento intracelular e citotoxicidade por microscopia. Notavelmente, os agentes antimicrobianos podem demonstrar sinergia em que o efeito combinado de dois ou mais agentes antimicrobianos, quando aplicados em conjunto seja maior do que quando aplicados separadamente. Teste para sinergia in vitro contra patógenos intracelulares é normalmente uma tarefa prodigiosa como permutações combinatórias de antibióticos em diferentes concentrações, devem ser avaliados. No entanto, descobrimos que nosso ensaio em tempo real combinada com automatizada, dispensando tecnologia digital permitted testes sinergia fácil. Usando estas abordagens, que foram capazes de levantamento sistematicamente acção de um grande número de agentes antimicrobianos sozinho e em combinação contra o patogénio intracelular, Legionella pneumophila.
Materials
| Name | Company | Catalog Number | Comments |
| J774A.1 cells | American Type Culture Collection | TIB-67 | Host cell |
| ACES | Sigma-Aldrich | A9758 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Yeast extract, ultrafiltered | Becton-Dickinson/Difco | 210929 | For making buffered charcoal yeast extract agar and buffered yeast extract medium; lower grades may cause impaired growth and/or alter sensitivity of Legionella to growth inhibitors |
| Alpha-ketoglutaric acid, monopotassium salt | Sigma-Aldrich | K2000 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Sodium pyruvate | Sigma-Aldrich | P5280 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Potassium phosphate, dibasic | Thermo Fisher Scientific | P288-500 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| L-cysteine | Sigma-Aldrich | C-7755 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Ammonium iron(III) citrate | Sigma-Aldrich | F5879 | For making buffered charcoal yeast extract agar and buffered yeast extract medium; ferric pyrophosphate may be used instead but is more difficult to weigh accurately |
| Potassium hydroxide solution, concentrated | Thermo Fisher Scientific | SP236-500 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Deonized water | N/A | N/A | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Thymidine (tissue culture grade) | Sigma-Aldrich | T1895 | For supplementing both RPMI 1640 and buffered yeast extract agar/medium — lower grade thymidine may be used for the latter, but may cause impaired cell growth and/or cell death in RPMI 1640 |
| RPMI 1640, standard formulation | Corning via Thermo Fisher Scientific | 10-040-CV | For growing J774A.1 cells prior to plating; includes 2 mM L-glutamine |
| RPMI 1640 lacking phenol red | Corning via Thermo Fisher Scientific | 17-105-CV | For plating J774A.1 cells in 384 well dishes (not suitable for growth prior to plating); also lacks L-glutamine — supplement to 2 mM before use |
| L-glutamine, 200 mM in 0.85% NaCl (tissue culture grade) | HyClone via Thermo Fisher Scientific | SH30034.02 | For supplementing RPMI 1640 lacking L-glutamine, to 2 mM final concentration |
| Iron-supplemented calf serum | Gemini Bioproducts | 100-510 | For supplementing RPMI 1640, to 9.1% final concentration |
| Trypan Blue solution | Sigma-Aldrich | T8154 | For staining for J774A.1 cell death determination while counting cell density |
| SYTOX Green, 5 mM solution in DMSO | Thermo Fisher Scientific | S7020 | For staining for J774A.1 cell death determination by fluorescence reading or epifluorescence microscopy (in conjunction with orange-red or far red fluorescent bacteria). Use at 125 nM final concentration. |
| Cell culture incubator | Thermo Fisher Scientific | 13-255-26 | For incubation of J774A.1 cells (both before and after infection); can also be used for incubation of bacteria, or a standard atmosphere incubator can be used instead) |
| Orbital shaker | BellCo Glass | 7744-01010 | For shaking incubation of J774A.1 cells before infection; fits inside cell culture incubator; includes shaker base 7744-01000 and tray 7740-01010 (these are also available separately) |
| Shaker flasks (250 ml) | ChemGlass Life Sciences | CLS-2038-04 | For shaking incubation of J774A.1 cells before infection |
| Shaker clamps for flasks (250 ml) | BellCo Glass | 7744-16250 | For shaking incubation of J774A.1 cells before infection |
| Shaker flasks (1,000 ml) | ChemGlass Life Sciences | CLS-2038-07 | For shaking incubation of J774A.1 cells before infection |
| Shaker clamps for flasks (1,000 ml) | BellCo Glass | 7744-16100 | For shaking incubation of J774A.1 cells before infection |
| Sponge foam caps for flasks (250 ml-1,000 ml) | ChemGlass Life Sciences | CLS-1490-038 | For shaking incubation of J774A.1 cells before infection; reduces risk of contamination relative to standard metal caps |
| MultiDrop Combi programmable multichannel peristaltic pump | Thermo Fisher Scientific | 5840300 | For dispensing J774A.1 cells, medium, and bacterial suspension containing fluorophores to large numbers of 384 well dishes |
| Combi standard bore manifold | Thermo Fisher Scientific | 24072670 | Default predispense volume of 20 μl is insufficient to compensate for settling — increase to 80 μl |
| White 384 well dishes treated for tissue culture | Corning | 3570 | For reading luminescence and fluorescence; Greiner catalog # 781080 also tested successfully |
| DMSO (tissue culture grade, in sealed ampoules) | Sigma-Aldrich | D2650 | For dissolving positive control and test compounds |
| Azithromycin | Sigma-Aldrich | PHR1088 | Antibiotic positive control |
| Saponin (from Quillaja bark) | Sigma-Aldrich | S-4521 | Cytoxicity positive control |
| Multichannel pipettor | Thermo Fisher Scientific | Finnpipette | For transfer of fixed amounts of positive control compounds; pipettor must have digital dispensing with detents to enable repetitive fixed volume dispensing |
| Epson pin transfer robot | Epson/ICCB-L | (Custom equipment) | For transfer of fixed amounts of test compounds from library arrays |
| D300 digital dispensing system | Hewlett-Packard via Tecan | D300 | For transfer of variable amounts of test compounds ranging from 11 picoliters to 10 µl |
| T8+ cartridges for D300 digital dispensing system | Hewlett-Packard via Tecan | T8+ | For dispensing test compounds |
| Epifluorescence microscope with computer-connected digital camera | Nikon | Ti | For live cell imaging; any standard fluorescent microscope can substitute, with phase contrast or DIC optics, capable of imaging green (fluorescein), orange-red to red (Texas Red), and far-red (Cy5) fluorescence, with 100X oil objective for highest resolution |
| Glass-bottom tissue culture dishes | MatTek Corporation | P35G-1.5-20-C | For live cell imaging. Dishes such as the MatTek allow microscopic visualization at 600X or 1,000X magnification through use of an inverted epifluorescent or confocal microscope. These specific dishes are 3.5 cm nominal diameter, 3.3 cm inside diameter, with 20 mm diameter #1.5 thickness cover slips inserted into the bottoms. |
| Photoshop CS6 | Adobe | Adobe photoshop or similar programs can be used to pseudocolor and merge light microscopic and fluorescent images. | |
| Mathematica 10 | Wolfam | For generation of two-dimensioonal isocontour isobolograms and three-dimensional surface isobolograms. |
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