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Fluorescent Detection of Intracellular Nitric Oxide in Staphylococcus aureus
荧光检测葡萄球菌属中的胞内一氧化氮   

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Abstract

Nitric Oxide (NO) is a highly-reactive radical gas that can modify a variety of cellular targets in both eukaryotes and bacteria. NO is produced endogenously by a wide variety of organisms: For example, as a cell-signaling molecule in mammals and bacteria via nitric oxide synthase (NOS) enzymes, and as a product of denitrification. As such, it is of great benefit to NO researchers to be able to sensitively detect intracellular NO and stable reactive nitrogen species (RNS) derived from NO. To this end, a protocol for fluorescent detection of intracellular NO/RNS in biofilm cultures of the Gram-positive pathogen Staphylococcus aureus has been optimized using the commercially-available cell-permeable fluorescent stain 4-Amino-5-Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM diacetate). This compound diffuses into cells and intracellular cleavage by esterase enzymes liberates weakly-fluorescent DAF-FM, which reacts with NO or other specific RNS to become highly fluorescent (Kojima et al., 1999). Although quantification of fluorescence is performed using a fluorescent plate reader, it is envisioned that this protocol could be adapted for intracellular NO/RNS imaging of S. aureus biofilms by confocal microscopy. Likewise, this technique could be optimized for the detection of intracellular NO/RNS in other growth conditions (i.e., planktonic cultures) and/or in other bacteria/archaea.

Materials and Reagents

  1. Wrapping film (Fisher Scientific, Parafilm MTM, catalog number: S37440 )
  2. Micro-centrifuge tubes (1.7 ml) (Fisher Scientific, catalog number: 14-222-171 )
  3. Sterile plastic culture tubes (Fisher Scientific, catalog number: 14-956-6A )
  4. Costar 3524 plates (24-well tissue culture treated) (Fisher Scientific, CorningTM CostarTM ,catalog number: 07-200-84 )
  5. Costar 3904 plates (96-well black tissue-culture treated) (Fisher Scientific, CorningTM, catalog number: 07-200-588 )
  6. 150-ml Nalgene sterile disposable 0.2 µm filter unit (Fisher Scientific, Thermo ScientificTM NalgeneTM, catalog number: 09-741-01 )
  7. 500-ml Nalgene sterile disposable 0.2 µm filter unit (Fisher Scientific, Thermo ScientificTM NalgeneTM, catalog number: 09-741-02 )
  8. S. aureus stock culture, stored at -80 °C in 25% (v/v) sterile glycerol
  9. Glycerol (Fisher Scientific, catalog number: G33-1 )
  10. Tryptic Soy Agar (TSA) plates (BD, BBLTM, catalog number: 221283 )
  11. Tryptic Soy Broth (TSB) (BD, DifcoTM, catalog number: 211825 )
  12. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-1 )
  13. Dextrose (Fisher Scientific, catalog number: D16-500 )
  14. 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM diacetate) (Thermo Fisher Scientific, catalog number: D-23842 )
  15. Dimethyl sulfoxide (DMSO) (100 ml) (Sigma-Aldrich, catalog number: 276855 )
  16. Human Plasma, lyophilized (5 ml) (Sigma-Aldrich, catalog number: P9523 )
  17. Carbonate-Bicarbonate buffer capsules (Sigma-Aldrich, catalog number: C3041 )
  18. 1x Hank's buffered salt solution (HBSS) buffer, containing calcium and magnesium (Fisher Scientific, CorningTM cellgroTM, catalog number: MT21023CV )
  19. Diethylamine (DEA) (100 ml) (Sigma-Aldrich, catalog number: 471216 )
  20. Diethylamine NONOate (DEA/NO) (10 mg vial) (Cayman Chemicals, catalog number: 82100 )
  21. 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide, monopotassium salt (cPTIO) (5 mg vial) (Cayman Chemicals, catalog number: 81540 )
  22. Sterile nuclease-free H2O (Fisher Scientific, InvitrogenTM AmbionTM, catalog number: AM9930 )
  23. NaOH (Thermo Fisher Scientific, catalog number: S318-500 )
  24. 50% (v/v) Glycerol Stock Solution(see Recipes)
  25. Biofilm Media (see Recipes)
  26. Carbonate-Bicarbonate Buffer (see Recipes)
  27. 20% Human Plasma (see Recipes)
  28. DAF-FM diacetate stock solution (see Recipes)
  29. 0.01 M NaOH solution (see Recipes)
  30. DEA/NO stock solution (see Recipes)
  31. cPTIO stock solution (see Recipes)
  32. DAF-FM diacetate working solution (see Recipes)
  33. DEA/NO working solution (see Recipes)
  34. DEA working solution (see Recipes)

Equipment

  1. Multi detection microplate reader (Biotek synergy HT, model: SIAFR )
  2. 10S Bio UV/Vis Spectrophotometer (Thermo Fisher Scientific, GENESYSTM, model: 840-208100 )
  3. Plate incubator (VWR International, catalog number: 97058-224 , model: 1556)
  4. Shaking incubator (VWR International, SignatureTM, catalog number: 14004-300 , model: 1570)
  5. Pipet-Lite XLS+ single-channel pipettes (2-20 µl, 20-200 µl, 100-1,000 µl) (Rainin, catalog number: 17014406 )
  6. Rack LTS tips (“P20” 2-20 µl, “P200” 20-200 µl, “P1000” 100-1,000 µl) (Rainin, Green-PakTM and SpaceSaverTM, catalog number: 17001865 , 17001863 and 17001864 )
  7. Labconco Class II A2 Biosafety Cabinet (Fisher Scientific, LabconcoTMPurifierTMLogic+TM, catalog number: 30-238-1100 )
  8. Vortex Mixer (Fisher Scientific, Fisher ScientificTM, catalog number: 02-215-365 )
  9. Micro-centrifuge (Fisher Scientific, Fisher ScientificTM accuSpinTM, catalog number: 13-100-675 )
  10. Chemical Fume Hood
  11. Refrigerator (4 °C)
  12. Freezer (-20 °C)
  13. Freezer (-80 °C)

Procedure

General safety notes for entire experiment:

  1. When working with BSL-2 organisms such as S. aureus, all potential aerosol-generating steps should be performed in a Class II A2 biosafety cabinet to ensure user safety. An asterisk (*) has been placed beside each step below which should be performed in a biosafety cabinet to preserve aseptic technique and user safety.
  2. Please note that although the demonstrations presented in videos 1-4 were filmed at a lab bench (to facilitate clear videography), these steps should be performed under a Class II A2 Biosafety Cabinet when working with BSL-2 bacteria.
  3. Likewise, MSDS sheets for DEA/NO, DEA, and DMSO (available online from the manufacturers) should be read carefully before starting this experiment, and recommended safety precautions be taken to avoid skin exposure and respiratory inhalation as necessary for each chemical. Therefore, perform chemical manipulations as appropriate under a chemical fume hood. Additionally, wear a lab coat, goggles, and nitrile gloves for all steps of the protocol, to comply with both biosafety and chemical safety considerations.

Day 1:


  1. *Streak a TSA plate for isolation from frozen glycerol stock of a S. aureus strain of interest. Incubate in plate incubator at 37 °C for 24 h. In this example protocol the clinical methicillin-susceptible strain UAMS-1 (Gillaspy et al., 1995) was used.

Day 2:

  1. *Use a single S. aureus colony (from TSA plate in step 1) to inoculate 3 ml of biofilm media in a sterile plastic culture tube. Grow in shaking incubator at 37 °C, 250 rpm for 16 h.
  2. *Add 350 µl 20% (v/v) human plasma solution to 4 wells of a Costar 24-well tissue culture plate. Seal the lid to the plate with wrapping film, and store at 4 °C until use on Day 3.

Day 3:

  1. Remove 24-well plate from 4 °C and equilibrate to room temperature.
  2. Determine the optical density at 600 nm (OD600) of a 20-fold dilution in sterile TSB of the S. aureus overnight culture using a spectrophotometer. Use sterile TSB as a blank control for the spectrophotometer. Multiply this reading by 20 to calculate the actual OD600/ml of the overnight culture.
  3. Calculate how much overnight culture to use to inoculate 1 ml biofilm media to a final OD600/ml = 0.05. Scale-up the volumes as necessary depending on the number of biofilm wells you plan to inoculate, and add 1 ml to account for pipetting error (In this particular sample protocol, 3 biofilm wells will be inoculated, therefore 4 ml of biofilm media containing diluted overnight culture is needed.).
  4. Using the P1000 pipette, carefully withdraw all plasma solution from each well of the 24-well plate. Tip the plate at a 15-30° angle and pipette from the corner of each well to ensure complete removal of the plasma solution.
  5. *Mix the diluted overnight culture (in this example 4 ml of diluted overnight culture in biofilm medium) by vortexing for 5 sec at top speed, and immediately transfer 1 ml to 3 plasma-coated wells from step 7.
  6. *Transfer 1 ml of sterile biofilm media to the forth (empty) plasma-coated well. This will serve as a negative control for aseptic technique.
  7. Place 24-well tissue culture plate in 37 °C plate incubator and grow for 7 h.
  8. Following growth, visually inspect the negative control well for contamination. The negative control well should look identical to the appearance of sterile media (i.e., no turbid growth or particulate matter should be present). If contamination occurs in the negative control well, do not proceed with the rest of the experiment.
  9. *Harvest the total biomass (biofilm + supernatant) from each well by vigorously pipetting, mixing, and scraping the bottom of the well with the P1000 pipette. This step usually takes 30 sec-1 min for each well, depending on the biofilm thickness. Performing work in a sterile environment is not necessary for sample integrity at this and subsequent steps. However, they should be performed under a Biosafety cabinet when working with BSL-2 organisms. Appropriate care should also be used to minimize cross-contamination of samples. Please refer to Video 1 for an example of this step. Transfer contents of each well to a sterile 1.7 ml micro-centrifuge tube.

    Video 1. Demonstration of biofilm harvesting from 24-well tissue culture plate

  10. Collect cells by centrifugation at 17,000 x g for 5 min at room temperature.
  11. While samples are centrifuging in step 13, dim the lights, remove a 5 µl aliquot of 5 mM DAF-FM diacetate stock solution from the -20 °C freezer, and thaw by vortexing.
    Note: DAF-FM is very light sensitive so be sure to perform all subsequent steps with the lights lowered. Dim ambient light (i.e., natural light from windows) is acceptable as long as the DAF-FM is not directly exposed to the light source.
  12. Once thawed, prepare the DAF-FM diacetate working solution. The working solution of DAF-FM diacetate should be wrapped in foil until used in step 17 below. This protocol works best if the 5 µM DAF-FM diacetate solution is prepared just prior to use in step 17 below.
  13. *Completely remove the culture supernatant from each centrifuged tube (step 13) with the P1000 pipette (Video 2).

    Video 2. Demonstration of supernatant removal from centrifuged cell pellet

  14. *Resuspend each cell pellet in 1 ml of 5 µM DAF-FM diacetate (prepared in step 15). S. aureus cell pellets will not resuspend well by vortexing alone, so use the P1000 pipette to break apart the cell pellet by scraping and pipetting (Video 3), then vortex the tube at top speed for 10 sec.

    Video 3. Demonstration of cell pellet resuspension in DAF-FM diacetate solution

  15. Incubate all tubes for 60 min at 37 °C in the plate incubator. Tubes may be covered in foil to reduce exposure to ambient light if this is a concern.
  16. While cell suspensions are incubating in step 18, freshly prepare the following solutions: 100 µM DEA in 1x HBSS, 100 µM DEA/NO in 1x HBSS.
  17. Collect cell pellets by centrifugation for 5 min, 17,000 x g at room temperature.
  18. *Discard supernatants as described in step 16 and Video 2.
  19. *To wash residual extracellular stain from cell pellets, resuspend each pellet in 1 ml 1x HBSS buffer as described in step 17 and Video 3.
  20. Collect cell pellets by centrifugation for 5 min, 17,000 x g at room temperature.
  21. *Discard supernatants as described in step 16 and Video 2.
  22. *Resuspend each cell pellet (as described in step 17 and Video 3) as follows:
    1. 1 pellet in 0.65 ml 1x HBSS buffer alone (“Untreated” cells stained with DAF-FM diacetate; baseline level of intracellular NO/RNS)
    2. 1 pellet in 0.65 ml 1x HBSS containing 100 µM DEA (“DEA” treated cells stained with DAF-FM diacetate; a control for the DEA portion of the chemical NO donor used in step 25c below)
    3. 1 pellet in 0.65 ml 1x HBSS containing 100 µM DEA/NO (“DEA/NO”-treated cells stained with DAF-FM diacetate; a positive control which should yield high-level intracellular fluorescence relative to the untreated and DEA treated samples)
    Note: It is important at this step that the sample cell densities are very similar to each other. In our experience, small variations in OD600 (i.e., sample OD600 values that are within 10% of each other) can be accounted for by reporting the data as RFU/OD (as described in step 28 below). However, larger variations in OD600 between samples will lead to inconsistent/difficult to interpret results. It is therefore recommended that the OD600 be checked at this step, and sample volumes adjusted accordingly, when working with different growth conditions or with biological samples that are suspected to grow at different rates during the initial culture incubation (step 10). For S. aureus samples, we typically aim for an OD600 ~1.0 per well. However, we have also observed reproducible results with well OD600 values as high as 2.0, again as long as the OD600 values of all samples across a given experiment are very similar to each other.
  23. *Immediately transfer 200 µl aliquots of each cell suspension in triplicate to wells of a Costar 3904 96-well plate (Video 4 and Figure 1). Additionally, transfer 200 µl aliquots of 1x HBSS in triplicate to this 96-well plate (This “buffer only” samples serves as a negative control for any background fluorescence attributable to the buffer itself. 1x HBSS tends to have low auto-fluorescence relative to cell samples.). Plate may be covered in foil to reduce exposure to ambient light at this step if this is a concern.

    Video 4. Demonstration of sample loading into 96-well plate


    Figure 1. Schematic of DAF-FM stained cell samples loaded in triplicate in a 96-well plate (Step 26). In this example experiment, higher-level DAF-FM fluorescence is expected in the positive control cell sample treated with DEA/NO (chemical NO donor; dark green wells), whereas detection of endogenous intracellular NO/RNS by DAF-FM fluorescence in the DEA treated and untreated samples (light green wells) are expected to be similar to each other, and both lower than the NO-treated positive control sample. Buffer alone (no DAF-FM stain present; white wells) should have very low levels of auto-fluorescence relative to the DAF-FM stained cells samples.

  24. Incubate this 96-well plate in the Biotek Synergy HT fluorescent plate reader. Time-course protocol settings should include the following: 37 °C, 3 sec medium shake prior to each reading, Fluorescence (EX/EM 485 ± 10/516 ± 10) and OD600 measurements recorded every 15 min for up to 60 min total. For S. aureus samples, the peak fluorescence of each sample usually occurs by 30 min.
  25. Report data as relative fluorescent units (RFU) per OD600 of each well. Please see (Lewis et al., 2015; Sapp et al., 2014) and Figure 2 for representative data.


    Figure 2. Detection of intracellular NO/RNS in S. aureus with DAF-FM diacetate. Cells harvested from replicate UAMS-17 h static biofilms were resuspended in 1x HBSS containing 5 µM DAF-FM diacetate. After incubation for 1 h at 37 °C, cells were collected by centrifugation, washed, and resuspended in 1x HBSS alone (“untreated”) or 1x HBSS supplemented with 100 µM DEA or 100 µM DEA/NO. Aliquots (200 µl) of each cell suspension were immediately transferred to a 96-well plate, and incubated at 37 °C in a Synergy HT fluorescent plate reader. Fluorescence and OD600 measurements were recorded after 30 min, and data were reported as relative fluorescent units (RFU) per OD600 of each well. Data represents the average of n = 3 independent experiments, error bars = SEM. *statistical significance compared to untreated UAMS-1 (P < 0.05, Tukey Test). The dataset used in this figure was originally published in (Sapp et al., 2014).

Notes

  1. This protocol is most reproducible when analyzing a small set (3-6) of samples per experiment. In our experience, scaling up the experiment to more than 6 samples tends to result in more variability between experiments. Although we have not dissected the reasons for this in detail, it is possible that the increased sample processing time required for more than 6 samples lends to increased variability in the exposure times of the samples to DAF-FM diacetate staining (steps 17-18) and/or to subsequent treatment steps (i.e., exposure of cell samples to DEA/NO as described in step 25), which could in turn influence the timing and level of DAF-FM fluorescence that occurs in the samples in response to NO. It is also possible that increased sample processing times may promote more variability in the exposure of the DAF-FM diacetate to ambient light. Variability between experiments can also minimized by ensuring that other biological variables (i.e., growth time of overnight cultures and biofilms) are kept consistent between experiments.
  2. Experiments should be repeated 3-6 times for adequate power for statistical analysis. In our experience the data tends to follow non-normal distribution, thus non-parametric tests are used to analyze the data for significance.
  3. Although DAF-FM can also react with certain RNS such as nitrosonium ions, the majority of the fluorescent signal has been shown to be due to NO (Kojima et al., 1998a; Kojima et al., 1998b). Since the NO radical itself is relatively unstable and may quickly yield other RNS upon exposure to cellular components, intracellular DAF-FM fluorescence should be considered an indirect measurement of NO levels.
  4. As an additional control, 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide, monopotassium salt (cPTIO), an NO scavenger, can be added to S. aureus cell suspensions to a concentration of 150 µM during the 1 h DAF-FM diacetate staining step (step 17).
  5. Although this protocol was originally developed to detect intracellular NO in S. aureus biofilm cultures, we have also used it with slight modifications to assess S. aureus intracellular NO levels in cells harvested from planktonic cultures and from agar plates (Sapp et al., 2014). However, when developing this assay for other bacterial or archaeal species, the DAF-FM diacetate concentration used, staining incubation time (step 17), and timing/frequency of fluorescent measurements (step 27) will require careful optimization. Per the manufacturer’s instructions, it is suggested that a concentration range of 1 µM-10 µM DAF-FM diacetate and 15-60 min staining time be initially tested to determine the optimal parameters for the experiment. According to the manufacturer’s protocol, it may take an additional 15-30 min after the initial DAF-FM staining step to allow complete de-esterification of the intra-cellular diacetates and liberation of intracellular DAF-FM. It is also recommended to run the appropriate DEA/NO and cPTIO controls when optimizing this assay. If results under optimized assay conditions are reproducible, these controls may be omitted from future experiments in order to be able to process more “unknown” samples per experiment. As well, the incubation time and/or frequency of RFU/OD600 data collection in the fluorescent plate reader may need to be extended or shortened, depending on the experimental growth conditions, microorganism being tested, and when the earliest “peak” DAF-FM fluorescence occurs.

Recipes

  1. 50% (v/v) Glycerol Stock Solution
    Mix 50 ml of glycerol with 50 ml deionized H2O. Filter-sterilize using a 150-ml Nalgene sterile disposable 0.2 µm filter unit or sterilize by autoclaving (liquid cycle for 30 min).
  2. Biofilm Media
    Dissolve 3 g TSB, 3 g NaCl, and 0.5 g dextrose in 100 ml deionized H2O. Transfer to a 200 ml glass media bottle and autoclave on liquid cycle for 20-25 min. Prepare freshly-made media for each weekly experiment(s). Biofilm media > 1 week old tends to lead to increased variation in biofilm growth.
  3. Carbonate-Bicarbonate Buffer
    Empty the contents of one carbonate-bicarbonate capsule into 100 ml of deionized H2O and dissolve. The contents of one capsule yields 100 ml of 0.05 M carbonate-bicarbonate buffer, pH 9.6 at 25 °C. Filter sterilize using a 150-ml Nalgene sterile disposable 0.2 µm filter unit.
  4. 20% Human Plasma
    Add 20 ml carbonate-bicarbonate directly to 5 ml lyophilized human plasma in its original vial. Mix gently for a few minutes to dissolve. This solution will appear as a translucent pale yellow-brown color. Store at 4 °C.
  5. DAF-FM diacetate stock solution
    Per the manufacturer’s instructions, prepare a 5 mM stock solution of DAF-FM diacetate (MW = 496) by dissolving (vortex to dissolve) the 1 mg packaging (D-23841) in 0.4 ml of high-quality anhydrous DMSO. Once dissolved in DMSO, the DAF-FM diacetate stock solution does not tolerate repeated freeze-thawing. Therefore, aliquot the stock solution into convenient one-time use working volumes (5-10 µl) into sterile 1.7 ml micro-centrifuge tubes pre-chilled on ice. Label tubes and immediately store in a light-tight container at -20 °C.
  6. 0.01 M NaOH solution
    Prepare a 0.01 M NaOH solution by dissolving 200 mg NaOH (FW = 40 g/mol) in 500 ml deionized H2O and dissolve. Filter sterilize using a 500-ml Nalgene sterile disposable 0.2 µm filter unit.
  7. DEA/NO stock solution
    Per the manufacturer’s instructions, prepare a 100 mM stock solution by dissolving a 10 mg vial of DEA/NO (FW = 206.3) into 323.2 µl of 0.01 M NaOH by vortexing. Once in solution, DEA/NO does not tolerate repeated freeze-thawing. Therefore, immediately aliquot as small working volumes (20-50 µl) into sterile 1.7 ml micro-centrifuge tubes pre-chilled on ice, and store at -80 °C. Do not use DEA/NO stock solution if it has been stored for more than two weeks at -80 °C.
  8. cPTIO stock solution
    Prepare a 150 mM stock solution of cPTIO (FW = 315.4 g/mol) by dissolving entire 5 mg contents of a cPTIO vial in 105.7 µl sterile nuclease-free H2O. Mix well by vortexing. This should be prepared fresh for each experiment during step 15 of this protocol. Per the manufacturer’s instructions, aqueous solutions of cPTIO are not stable for more than one day.
  9. DAF-FM diacetate working solution
    Prepare a 5 µM working solution of DAF-FM diacetate by diluting a thawed aliquot of 5 mM DAF-FM stock solution 1,000-fold into 1x HBSS buffer. Mix well by vortexing. This should be prepared fresh for each experiment during step 15 of this protocol.
  10. DEA/NO working solution
    Prepare a 100 µM working solution by adding 3.33 µl DEA/NO stock solution to 5 ml of 1x HBSS. This should be prepared fresh for each experiment during step 19 of this protocol.
  11. DEA working solution
    First, prepare a 100 mM stock solution of DEA by adding 51.7 µl DEA (0.707 g/ml density, FW = 73.14 g/mol) to 5 ml 1x HBSS. Then, dilute this 1,000-fold in 1x HBSS to a 100 µM working solution. This should be prepared fresh for each experiment during step 19 of this protocol.

Acknowledgments

This work was funded in part by resubmission funding from the University of Florida Emerging Pathogens Institute, a University of Florida IFAS Early Career Award, and NIH grant AI118999, all to KCR. This protocol and the dataset depicted in Figure 2 have both been adapted from (Sapp et al., 2014), and is reproduced here under the Creative Commons Attribution (CC BY) license policy of PLOS ONE.

References

  1. Gillaspy, A. F., Hickmon, S. G., Skinner, R. A., Thomas, J. R., Nelson, C. L. and Smeltzer, M. S. (1995). Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis. Infect Immun 63(9): 3373-3380.
  2. Kojima, H., Nakatsubo, N., Kikuchi, K., Kawahara, S., Kirino, Y., Nagoshi, H., Hirata, Y. and Nagano, T. (1998a). Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 70(13): 2446-2453.
  3. Kojima, H., Sakurai, K., Kikuchi, K., Kawahara, S., Kirino, Y., Nagoshi, H., Hirata, Y. and Nagano, T. (1998b). Development of a fluorescent indicator for nitric oxide based on the fluorescein chromophore. Chem Pharm Bull (Tokyo) 46(2): 373-375.
  4. Kojima, H., Urano, Y., Kikuchi, K., Higuchi, T., Hirata, Y. and Nagano, T. (1999). Fluorescent Indicators for Imaging Nitric Oxide Production. Angew Chem Int Ed Engl 38(21): 3209-3212.
  5. Lewis, A. M., S. S. Matzdorf, J. L. Endres, I. H. Windham, K. W. Bayles, and K. C. Rice. (2015). Examination of the Staphylococcus aureus nitric oxide reductase (saNOR) reveals its contribution to modulating intracellular no levels and cellular respiration. Mol Microbiol 96: 651-69.
  6. Sapp, A. M., A. B. Mogen, E. A. Almand, F. E. Rivera, L. N. Shaw, A. R. Richardson, and K. C. Rice. (2014). Contribution of the nos-pdt operon to virulence phenotypes in methicillin-sensitive Staphylococcus aureus. PLoS One 9: e108868.

简介

一氧化氮(NO)是一种高反应性的自由基气体,其可以修饰真核生物和细菌中的多种细胞靶标。 NO由多种生物体内源性产生:例如,作为哺乳动物和细菌中的细胞信号分子,通过一氧化氮合酶(NOS)酶,以及作为脱氮的产物。因此,NO研究人员能够敏感地检测来自NO的细胞内NO和稳定的活性氮物质(RNS)是非常有益的。为此,已经使用商业上可获得的细胞可渗透的荧光染料4-氨基-5来优化用于荧光检测革兰氏阳性病原体金黄色葡萄球菌的生物膜培养物中的细胞内NO/RNS的方案 - 甲基氨基-2',7'-二氟荧光素二乙酸酯(DAF-FM二乙酸酯)。该化合物扩散到细胞中并且通过酯酶的细胞内裂解释放弱荧光DAF-FM,其与NO或其它特异性RNS反应以变成高度荧光的(Kojima等人,1999)。虽然使用荧光板读数器进行荧光的定量,但设想该方案可适用于S的细胞内NO/RNS成像。 aureus biofilms by confocal microscopy。同样,这种技术可以被优化用于检测其它生长条件(即浮游生物培养物)和/或其它细菌/古细菌中的细胞内NO/RNS。

材料和试剂

  1. 包装膜(Fisher Scientific,Parafilm M ,目录号:S37440)
  2. 微量离心管(1.7ml)(Fisher Scientific,目录号:14-222-171)
  3. 无菌塑料培养管(Fisher Scientific,目录号:14-956-6A)
  4. Costar 3524平板(24孔组织培养处理的)(Fisher Scientific,Corning,TM Costar,sup。TM,目录号:07-200-84)
  5. Costar 3904板(96孔黑色组织培养处理)(Fisher Scientific,Corning公司,目录号:07-200-588)
  6. 150-ml Nalgene无菌一次性0.2μm过滤单元(Fisher Scientific,Thermo Scientific Nalgene TM ,目录号:09-741-01)
  7. 500-ml Nalgene无菌一次性0.2μm过滤单元(Fisher Scientific,Thermo Scientific Nalgene TM,目录号:09-741-02)

  8. 金黄色葡萄球菌储存培养物,在-80℃下在25%(v/v)无菌甘油中储存。
  9. 甘油(Fisher Scientific,目录号:G33-1)
  10. 胰蛋白酶大豆琼脂(TSA)板(BD,BBL TM ,目录号:221283)
  11. 胰蛋白酶大豆肉汤(TSB)(BD,Difco TM ,目录号:211825)
  12. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-1)
  13. 右旋糖(Fisher Scientific,目录号:D16-500)
  14. 4-氨基-5-甲基氨基-2',7'-二氟荧光素二乙酸酯(DAF-FM二乙酸酯)(Thermo Fisher Scientific,目录号:D-23842)
  15. 二甲基亚砜(DMSO)(100ml)(Sigma-Aldrich,目录号:276855)
  16. 人血浆,冻干(5ml)(Sigma-Aldrich,目录号:P9523)
  17. 碳酸盐 - 碳酸氢盐缓冲液胶囊(Sigma-Aldrich,目录号:C3041)
  18. 含有钙和镁的1×Hank's缓冲盐溶液(HBSS)缓冲液(Fisher Scientific,Corning公司,目录号:MT21023CV)
  19. 二乙胺(DEA)(100ml)(Sigma-Aldrich,目录号:471216)
  20. 二乙胺NONOate(DEA/NO)(10mg小瓶)(Cayman Chemicals,目录号:82100)
  21. (cPTIO)(5mg小瓶)(Cayman Chemicals,Inc。),将2-(4-羧基苯基)-4,5-二氢-4,4,5,5-四甲基-1H-咪唑基-1-氧-3-氧化物,单钾盐目录号:81540)
  22. 无菌无核酸酶H 2 O(Fisher Scientific,Invitrogen TM,目录号:AM9930)
  23. NaOH(Thermo Fisher Scientific,目录号:S318-500)
  24. 50%(v/v)甘油储备溶液(见配方)
  25. 生物膜介质(参见配方)
  26. 碳酸盐 - 碳酸氢盐缓冲液(参见配方)
  27. 20%人血浆(见配方)
  28. DAF-FM二乙酸酯储备溶液(见配方)
  29. 0.01 M NaOH溶液(见配方)
  30. DEA/NO储备溶液(见配方)
  31. cPTIO储备溶液(见配方)
  32. DAF-FM二乙酸酯工作溶液(参见配方)
  33. DEA/NO工作解决方案(参见配方)
  34. DEA工作解决方案(参见配方)

设备

  1. 多检测酶标仪(Biotek synergy HT,型号:SIAFR)
  2. 10S Bio UV/Vis分光光度计(Thermo Fisher Scientific,GENESYS TM ,型号:840-208100)
  3. 平板培养箱(VWR International,目录号:97058-224,型号:1556)
  4. 摇动培养箱(VWR International,Signature TM ,目录号:14004-300,型号:1570)
  5. Pipet-Lite XLS +单通道移液器(2-20μl,20-200μl,100-1,000μl)(Rainin,目录号:17014406)
  6. 将Rack LTS吸头("P20"2-20μl,"P200"20-200μl,"P1000"100-1000μl)(Rainin,Green-Pak TM sups和SpaceSaver TM sup- sup>,目录号:17001865,17001863和17001864)
  7. Labconco II类A2生物安全柜(Fisher Scientific,Labconco< sup> TM< sup> TM Logic + TM ,目录号:30-238-1100) >
  8. 涡旋混合器(Fisher Scientific,Fisher Scientific ,目录号:02-215-365)
  9. 微型离心机(Fisher Scientific,Fisher Scientific accuSpin TM ,目录号:13-100-675)
  10. 化学烟气罩
  11. 冰箱(4°C)
  12. 冷冻(-20°C)
  13. 冷冻(-80℃)

程序

整个实验的一般安全说明:
a。当使用BSL-2生物体如金黄色葡萄球菌时,所有潜在的气溶胶产生步骤应在II类A2生物安全柜中进行,以确保用户安全。在每个步骤旁边放置一个星号(*),应在生物安全柜中进行,以保持无菌技术和用户安全。
请注意,虽然在视频1-4中展示的演示在实验室工作台上拍摄(以便于清晰的视频摄影),但在使用BSL-2时,这些步骤应在II类A2生物安全柜下进行菌。
c。同样,在开始本实验之前,应仔细阅读DEA/NO,DEA和DMSO(可从制造商在线获取)的MSDS表,并采取建议的安全预防措施,以避免皮肤接触和呼吸吸入每种化学品都需要。因此,在化学通风橱内进行适当的化学操作。此外,为协议的所有步骤佩戴实验室外套,护目镜和丁腈手套,以符合生物安全和化学品安全考虑。

第1天:

  1. *划线用于从冷冻甘油储液分离的TSA板。金黄色葡萄球菌。在平板孵育器中在37℃孵育24小时。在本实施例方案中,使用临床甲氧西林敏感菌株UAMS-1(Gillaspy等人,1995)。

    第2天:
  2. *使用单个 S。金黄色葡萄球菌(来自步骤1中的TSA平板)以在无菌塑料培养管中接种3ml生物膜培养基。在振荡培养箱中在37℃,250rpm生长16小时。
  3. *向4孔Costar 24孔组织培养板中加入350μl20%(v/v)人血浆溶液。用包装膜将盖子盖在板上,并在4℃下保存,直到第3天使用。

    第3天:
  4. 从4℃取出24孔板,平衡至室温
  5. 测定在无菌TSB中20倍稀释的600nm处的光密度(OD 600)。 aureus 过夜培养物。使用无菌TSB作为分光光度计的空白对照。将该读数乘以20以计算过夜培养物的实际OD 600/ml/ml。
  6. 计算使用多少过夜培养物接种1ml生物膜培养基至最终OD 600/ml = 0.05。根据计划接种的生物膜孔的数量,根据需要放大体积,并添加1ml以解决移液误差(在该特定的样品方案中,将接种3个生物膜孔,因此4ml的含有稀释的生物膜培养基过夜培养)。
  7. 使用P1000移液管,小心地从24孔板的每个孔中取出所有血浆溶液。以15-30°的角度倾斜平板,从每个孔的角部移液,以确保完全去除血浆溶液。
  8. *通过以最高速度涡旋5秒,混合稀释的过夜培养物(在该实施例中,4ml在生物膜培养基中的稀释的过夜培养物),并立即将1ml转移到来自步骤7的3个涂覆有等离子体的孔中。
  9. *转移1毫升无菌生物膜介质到第四(空)血浆涂层井。这将作为无菌技术的阴性对照。
  10. 将24孔组织培养板置于37℃培养板中培养7小时
  11. 生长后,目视检查阴性对照孔的污染。阴性对照孔应该看起来与无菌培养基的外观相同(即不存在混浊生长或颗粒物质)。如果在阴性对照孔中发生污染,则不要继续实验的其余部分
  12. *通过用P1000移液管强力吸取,混合和刮下孔底部,从每个孔收获总生物量(生物膜+上清液)。根据生物膜厚度,该步骤通常需要每个孔30秒-1分钟。在这个和随后的步骤中,在无菌环境中进行工作不是样品完整性的必要条件。然而,当使用BSL-2生物体时,它们应当在生物安全柜下进行。还应当适当注意使样品的交叉污染最小化。有关此步骤的示例,请参阅视频1。将每个孔的内容物转移到无菌的1.7ml微量离心管中
    <! - flashid1878v76开始 - >
    视频1.来自24孔组织培养板的生物膜收获的演示
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  13. 通过在室温下以17,000×g离心5分钟收集细胞
  14. 当样品在步骤13中离心时,使灯变暗,从-20℃冰箱中取出5μl等分的5mM DAF-FM二乙酸酯储备溶液,并通过涡旋解冻。
    注意:DAF-FM非常光敏,因此请确保执行所有后续步骤与降低灯。只要DAF-FM不直接暴露于光源,昏暗的环境光(即来自窗口的自然光)是可接受的。
  15. 一旦解冻,准备DAF-FM二乙酸酯工作溶液。 DAF-FM二乙酸酯的工作溶液应该用箔包裹,直到在下面的步骤17中使用。如果在使用前在下面的步骤17中制备5μMDAF-FM二乙酸盐溶液,则该方案效果最好。
  16. *用P1000移液管(视频2)从每个离心管中完全除去培养物上清液(步骤13)。

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    视频2.从离心细胞沉淀中去除上清液的示例
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  17. *将每个细胞沉淀重悬在1ml5μMDAF-FM二乙酸酯(在步骤15中制备)中。


    金黄色葡萄球菌细胞沉淀不能单独通过涡旋重新悬浮,因此使用P1000移液管通过刮擦和移液分离细胞沉淀(视频3),然后以最高速度旋转管子10秒。

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    视频3.在DAF-FM二乙酸盐溶液中显示细胞沉淀重悬。
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  18. 孵育所有管60分钟,在37℃在板培养箱。管可以覆盖箔,以减少暴露于环境光,如果这是一个关注
  19. 当细胞悬浮液在步骤18中孵育时,新鲜制备以下溶液:100μMDEA在1x HBSS中,100μMDEA/NO在1x HBSS中。
  20. 通过在室温下离心5分钟收集细胞沉淀物,17,000×g
  21. *按照步骤16和视频2中所述弃去上清液。
  22. *要清洗细胞沉淀中的残留细胞外染色,按照步骤17和视频3所述,将每个沉淀重悬于1ml 1×HBSS缓冲液中。
  23. 通过在室温下离心5分钟收集细胞沉淀物,17,000×g
  24. *按照步骤16和视频2中所述弃去上清液。
  25. *如下所述重悬每个细胞沉淀(如步骤17和视频3中所述):
    1. ("用DAF-FM二乙酸酯染色的未处理"细胞;细胞内NO/RNS的基线水平)
    2. 1颗粒在含有100μMDEA(用DEAF-FM二乙酸酯染色的"DEA"处理的细胞;在下面步骤25c中使用的化学NO供体的DEA部分的对照)的0.65ml 1×HBSS中,
    3. 1颗粒在含有100μMDEA/NO(用DEA-FM二乙酸酯染色的"DEA/NO"处理的细胞;相对于未处理和DEA处理的样品应当产生高水平细胞内荧光的阳性对照)的0.65ml 1x HBSS中,
    注意:在此步骤中重要的是,样品池密度彼此非常相似。根据我们的经验,OD 600的小变化(即,彼此在10%以内的样品OD 600个值)可以通过将数据报告为RFU/OD(如下面步骤28所述)。然而,样品之间的OD 600的较大变化将导致不一致/难以解释结果。因此,建议在该步骤检查OD <600>,并且在使用不同的生长条件或怀疑在初始培养温育期间以不同速率生长的生物样品时,相应地调节样品体积(步骤10)。对于金黄色葡萄球菌样品,我们通常瞄准每孔OD 600?1.0。然而,我们还观察到具有高达2.0的良好OD 600值的可重复结果,同样只要给定实验中所有样品的OD 600值非常相似彼此。
  26. 立即将200μl等份的每种细胞悬浮液一式三份转移到Costar 3904 96孔板的孔中(视频4和图1)。另外,将200μl1×HBSS的等分试样一式三份转移到该96孔板(该"仅缓冲液"样品用作可归因于缓冲液本身的任何背景荧光的阴性对照)。1 HBSS倾向于具有相对于细胞样品)。如果这是一个需要考虑的问题,板可以覆盖在箔中以减少暴露在环境光下
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    视频4.将样品加载到96孔板中的示范
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    图1.在96孔板中一式三份装载的DAF-FM染色的细胞样品的示意图(步骤26)。在本实施例实验中,阳性对照中预期有更高水平的DAF-FM荧光用DEA/NO(化学NO供体;深绿色孔)处理的细胞样品,而在DEA处理和未处理样品(浅绿色孔)中通过DAF-FM荧光检测内源细胞内NO/RNS预期彼此相似,且均低于经NO处理的阳性对照样品。单独的缓冲液(不存在DAF-FM染色;白色孔)相对于DAF-FM染色的细胞样品应具有非常低的自发荧光水平。

  27. 将此96孔板在Biotek Synergy HT荧光板读数器中孵育。时间过程方案设置应包括以下:在每个读数之前37℃,3秒中等摇动,每15天记录荧光(EX/EM 485±10/516±10)和OD 600次测量min,总共60分钟。对于 s。 aureus样品,每个样品的峰值荧光通常发生30分钟
  28. 报告数据为每个孔的OD 600的相对荧光单位(RFU)。请参阅(Lewis等人,2015; Sapp等人,2014年)和图2的代表性数据。


    图2.检测细胞内NO/RNS。 aureus 与DAF-FM二乙酸酯。将从重复的UAMS-17h静态生物膜收获的细胞重悬于含有5μMDAF-FM二乙酸酯的1×HBSS中。在37℃孵育1小时后,通过离心收集细胞,洗涤,并重悬于单独的1×HBSS("未处理的")或补充有100μMDEA或100μMDEA/NO的1×HBSS中。将每个细胞悬浮液的等分试样(200μl)立即转移到96孔板中,并在Synergy HT荧光板读数器中在37℃下温育。在30分钟后记录荧光和OD 600测量,并且数据报告为每个孔的每OD 600个的相对荧光单位(RFU)。数据表示n = 3次独立实验的平均值,误差条= SEM。 *与未处理的UAMS-1相比的统计学显着性(p <0.05,Tukey试验)。该图中使用的数据集最初发布在(Sapp等人。,2014)中。

笔记

  1. 当分析每个实验的小组(3-6)样品时,该方案是最可重复的。根据我们的经验,将实验放大到超过6个样品往往导致实验之间更多的变异性。虽然我们没有详细解释其原因,但是对于多于6个样品所需的增加的样品处理时间可能增加样品对DAF-FM二乙酸酯染色的曝光时间的变化性(步骤17-18)和/或随后的处理步骤(即,如步骤25中所述将细胞样品暴露于DEA/NO),其可进而影响发生在DAF/FM荧光中的DAF-FM荧光的时间和水平样品响应NO。还有可能的是,增加的样品处理时间可以促进DAF-FM二乙酸酯暴露于环境光的更多变化性。通过确保其他生物变量(即,过夜培养物和生物膜的生长时间)在实验之间保持一致,实验之间的变异性也可以最小化。
  2. 实验应重复3-6次,以获得足够的功率进行统计分析。在我们的经验中,数据往往遵循非正态分布,因此非参数检验用于分析数据的重要性。
  3. 虽然DAF-FM也可以与某些RNS如亚硝鎓离子反应,但是大部分荧光信号已经显示是由于NO引起的(Kojima,Nakatsubo等人1998,Kojima,Sakurai, 等,1998)。由于NO基团本身相对不稳定,并且当暴露于细胞组分时可快速产生其它RNS,所以细胞内DAF-FM荧光应被认为是NO水平的间接测量。
  4. 作为另外的对照,将2-(4-羧基苯基)-4,5-二氢-4,4,5,5-四甲基-1H-咪唑基-1-氧-3-氧化物,单钾盐(cPTIO),NO清除剂,可以添加到 S。在1小时DAF-FM二乙酸酯染色步骤(步骤17)期间,将金黄色葡萄球菌细胞悬浮液浓缩至150μM。
  5. 虽然该协议最初被开发用于检测细胞内NO。金黄色葡萄球菌生物膜培养物,我们也使用它与轻微的修改来评估。在从浮游培养物和从琼脂平板收获的细胞中的金黄色葡萄球菌细胞内NO水平(Sapp等人,2014)。然而,当开发用于其它细菌或古细菌物种的该测定时,使用的DAF-FM二乙酸盐浓度,染色孵育时间(步骤17)和荧光测量的时间/频率(步骤27)将需要仔细优化。根据制造商的说明,建议最初测试1μM-10μMDAF-FM二乙酸酯的浓度范围和15-60分钟染色时间,以确定实验的最佳参数。根据制造商的方案,在初始DAF-FM染色步骤后可能需要额外的15-30分钟,以允许细胞内二乙酸酯的完全脱酯化和细胞内DAF-FM的释放。当优化该测定时,也建议运行合适的DEA/NO和cPTIO对照。如果在优化的测定条件下的结果是可重复的,则这些对照可以从未来的实验中省略,以便能够每个实验处理更多的"未知"样品。同样,取决于实验的生长条件,被测试的微生物,以及何时,可能需要延长或缩短荧光读板器中的RFU/OD 600数据收集的孵育时间和/或频率发生最早的"峰"DAF-FM荧光。

食谱

  1. 50%(v/v)甘油储备液
    将50ml甘油与50ml去离子H 2 O混合。使用150 ml Nalgene无菌一次性0.2μm过滤器过滤灭菌,或通过高压灭菌(液体循环30分钟)灭菌。
  2. 生物膜媒体
    将3g TSB,3g NaCl和0.5g葡萄糖溶解在100ml去离子H 2 O中。转移到200ml玻璃介质瓶和高压釜液体循环20-25分钟。为每周的实验准备新鲜制备的培养基。生物膜培养基> 1周龄往往导致生物膜生长的变化增加
  3. 碳酸盐 - 碳酸氢盐缓冲液
    将一个碳酸盐 - 碳酸氢盐胶囊的内容物倒入100ml去离子H 2 O中并溶解。一个胶囊的内容物在25℃下产生100ml的0.05M碳酸盐 - 碳酸氢盐缓冲液,pH 9.6。使用150-ml Nalgene无菌一次性0.2μm过滤器单元过滤灭菌
  4. 20%人血浆
    将20ml碳酸盐 - 碳酸氢盐直接加入其原始小瓶中的5ml冻干人血浆中。轻轻混合几分钟以溶解。该溶液将呈现为半透明的浅黄褐色。储存于4°C。
  5. DAF-FM二乙酸酯储备液
    根据制造商的说明,通过将1mg包装(D-23841)溶解(涡旋以溶解)在0.4ml高品质无水DMSO中,制备5mM DAF-FM二乙酸酯储液(MW = 496)。一旦溶解在DMSO中,DAF-FM二乙酸酯储备溶液不耐受反复的冻融。因此,将储备溶液分成方便的一次性使用工作体积(5-10μl)到在冰上预冷的无菌1.7ml微量离心管中。标签管,立即存放在-20°C的不透光容器中。
  6. 0.01 M NaOH溶液
    通过将200mg NaOH(FW = 40g/mol)溶解在500ml去离子H 2 O中制备0.01M NaOH溶液并溶解。使用500-ml Nalgene无菌一次性0.2μm过滤器单元过滤灭菌
  7. DEA/NO储备液
    根据制造商的说明,通过涡旋将10mg小瓶的DEA/NO(FW = 206.3)溶解在323.2μl的0.01M NaOH中制备100mM储备溶液。一旦在溶液中,DEA/NO不耐受反复的冻融。因此,立即等分作为小工作体积(20-50μl)到在冰上预冷的无菌的1.7ml微离心管中,并储存在-80℃。如果已经在-80℃下储存超过两周,则不要使用DEA/NO储备溶液
  8. cPTIO储液
    通过将整个5mg内容物的cPTIO小瓶溶解在105.7μl无菌无核酸酶H 2 O中制备150mM cPTIO储备溶液(FW = 315.4g/mol)。通过涡旋混合。在本方案的步骤15期间,应该为每个实验新鲜制备。根据制造商的说明,cPTIO的水溶液不稳定超过一天
  9. DAF-FM二乙酸酯工作溶液
    通过将解冻的5mM DAF-FM储备液的等分试样稀释1000倍至1x HBSS缓冲液中,制备5μM的DAF-FM二乙酸酯工作溶液。通过涡旋混合。在本协议的第15步中,应为每个实验准备新鲜的
  10. DEA /无工作溶液
    通过添加3.33微升DEA/NO储备溶液到5毫升1 HBSS中准备一个100μM工作溶液。在本协议的第19步中,应为每个实验准备新鲜的
  11. DEA工作溶液
    首先,通过向5ml 1x HBSS中加入51.7μlDEA(0.707g/ml密度,FW = 73.14g/mol)制备100mM DEA储备溶液。然后,在1x HBSS中将该1,000倍稀释至100μM工作溶液。在本协议的第19步中,应为每个实验准备新鲜的

致谢

这项工作的部分资金来自佛罗里达大学新兴病原体研究所,佛罗里达大学IFAS早期职业奖和NIH授予AI118999,所有到KCR的重新提交资金。该协议和图2中描绘的数据集都已经从(Sapp等人,2014)改编,并且在此根据PLOS ONE的知识共享署名(CC BY)许可策略被再现。

参考文献

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  2. Kojiima,H.,Nakatsubo,N.,Kikuchi,K.,Kawahara,S.,Kirino,Y.,Nagoshi,H.,Hirata,Y.and Nagano,T。(1998)。  用新型荧光指示剂检测和成像一氧化氮:二氨基荧光素。 Anal Chem 70(13):2446-2453
  3. Kojima,H.,Sakurai,K.,Kikuchi,K.,Kawahara,S.,Kirino,Y.,Nagoshi,H.,Hirata,Y.and Nagano,T。(1998)。  基于荧光素发色团的一氧化氮荧光指示剂的开发。 em> Chem Pharm Bull (Tokyo)46(2):373-375
  4. Koijima,H.,Urano,Y.,Kikuchi,K.,Higuchi,T.,Hirata,Y.and Nagano,T。(1999)。  用于成像一氧化氮生产的荧光指示剂。 Angew Chem Int Ed Engl 38(21): 3209-3212。
  5. Lewis,A.M.,S.S.Matzdorf,J.L.Endres,I.H.Windham,K.W.Bayles和K.C.Rice。 (2015)。  检查金黄色葡萄球菌一氧化氮还原酶(saNOR)显示其对调节细胞内无水平和细胞呼吸的贡献。 Mol Microbiol 96:651-69。
  6. Sapp,A.M.,A.B.Mogen,E.A.Almand,F.E.Rivera,L.N.Shaw,A.R.Richardson和K.C.Rice。 (2014)。  nos-pdt操纵子对在甲氧西林敏感的金黄色葡萄球菌中的毒力表型。 9:e108868。
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引用:Lewis, A. M., Matzdorf, S. S. and Rice, K. C. (2016). Fluorescent Detection of Intracellular Nitric Oxide in Staphylococcus aureus. Bio-protocol 6(14): e1878. DOI: 10.21769/BioProtoc.1878.
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