eoe sub articles

EoE Sub Articles

1. Evaluation of Antimicrobial Activity
NOTE: All work involving bacteria should be carried out under sterile conditions to avoid contamination of either the assay or the laboratory. All media should be autoclaved before use, and plasticware and equipment such as pipettes must be kept sterile. It is recommended that work be done in a biocontainment hood (type 2).
<ol>
	<li>Streak glycerol stocks of bacterial strains appropriate for the antibiotic scaffold onto lysogeny broth agar (LB, prepared per manufacturer&#39;s instructions), and grow overnight at 37 &#176;C. 
	NOTE: The choice of bacteria to test antibacterial activity must be made based on the antibiotic scaffold being used. A representative range of 5&#8211;10 bacteria should be chosen from the species that are known to be susceptible to the antibiotic, with consideration given to the logistical capabilities of the lab. If possible, resistant bacteria should also be tested. The protocol given below will work on most bacteria, but check if special conditions are required (e.g., CO2, special media) and make alterations as necessary. Bacteria successfully assayed using these conditions include Staphylococci, Streptococci, Bacilli, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterococcus faecium.</li>
	<li>Pick a single colony from the plate, and culture overnight in 5 mL of cation adjusted Mueller-Hinton Broth (CAMHB, prepared per manufacturer&#8217;s instructions) at 37 &#176;C.</li>
	<li>Dilute the overnight cultures ~40-fold in CAMHB and grow to a mid-log phase, optical density at 600 nm (OD600) = 0.4&#8211;0.8, volume 5 mL).</li>
	<li>Prepare stock solutions of each fluorescent antibiotic at 1.28 mg/mL in sterile water, and pipette 10 &#956;L of antibiotic to the first column of a 96 well plate.</li>
	<li>Add 90 &#956;L of CAMHB to the first column and 50 &#956;L to all other wells. Then, perform serial 2-fold dilution across the plate.</li>
	<li>Thoroughly mix, then dilute the mid-log phase cultures to ~106 colony forming units (CFU)/mL and add 50 &#956;L to all wells, to provide a final concentration of ~5 x 105 CFU/mL. 
	volume of culture (mL) = (media volume in mL)/(OD600 x 1,000) 
	e.g., for an OD600 = 0.5 culture in a desired media volume of 12 mL, add (12/(0.5 x 1,000) = 0.024 mL of culture to 12 mL of media</li>
	<li>Cover the plates with lids and incubate at 37 &#730;C for 18&#8211;24 h without shaking.</li>
	<li>Visually inspect the plates, with the MIC being the lowest concentration well with no visible growth. 
	NOTE: See Table 1 for some examples of active and inactive fluorescent antibiotics.</li>
</ol>
2. Analysis of Probe Accumulation by Spectrophotometry
NOTE: These centrifugation times have been optimized for E. coli, so slight alterations may be needed for other species. Representative data for probe accumulation is reported for the NBD-labeled ciprofloxacin probe.
<ol>
	<li>Streak glycerol stocks of the bacterial strains onto LB agar and grow overnight at 37 &#176;C.</li>
	<li>Pick a single colony from the plate and culture overnight in LB at 37 &#176;C.</li>
	<li>Dilute overnight cultures ~50-fold in media and grow to mid-log phase (OD600 = 0.4&#8211;0.8).</li>
	<li>Centrifuge the cultures at 1,470 x g for 25 min and decant the media.</li>
	<li>Resuspend the bacteria in 1 mL of phosphate-buffered saline (PBS), then centrifuge at 1,470 x g for 15 min.</li>
	<li>Decant the media and resuspend the washed pellets in PBS to a final OD600 = 2.</li>
	<li>If desired, add 10.1 &#956;L of carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 10 mM in PBS) to 1 mL of bacteria (final concentration 100 &#956;M) and incubate at 37 &#730;C for 10 min. 
	NOTE: CCCP is an efflux pump inhibitor. Addition of CCCP will allow the examination of the impact of efflux.</li>
	<li>Centrifuge the cultures at 18,000 x g for 4 min at 20 &#176;C and decant the media.</li>
	<li>Add 1 mL of fluorescent antibiotic solution (10&#8211;100 &#956;M in PBS) to the pellet, and incubate at 37 &#176;C for 30 min.</li>
	<li>Centrifuge the cultures at 18,000 x g for 7 min at 4 &#176;C and decant the media.</li>
	<li>Resuspend the bacteria in 1 mL of cold PBS, and repeat step 2.9.</li>
	<li>Repeat step 2.10 a total of 4x.</li>
	<li>If desired, lyse bacteria by adding 180 &#956;L of lysis buffer (20 mM Tris-HCl, pH 8.0, and 2 mM sodium EDTA) then 70 &#956;L of lysozyme (72 mg/mL in H2O).</li>
	<li>Incubate at 37&#176;C for 30 min, then freeze-thaw 3x (&#8722;78 &#176;C for 5 min, then 34 &#176;C for 15 min).</li>
	<li>Sonicate the samples for 20 min, then heat to 65 &#176;C for 30 min.</li>
	<li>Centrifuge the lysed samples (18,000 x g, 8 min), then filter through a 10 kDa filter membrane.</li>
	<li>Wash the filter the 4x with 100 &#956;L of water.</li>
	<li>Transfer the lysate to a flat-bottom black 96 well plate and measure the fluorescence intensity on a plate reader with excitation and emission wavelengths appropriate for the fluorophore (i.e., 7-(dimethylamino)-2-oxo-2H-chromen-4-yl [DMACA]: &#955;ex = 400 nm, &#955;em = 490 nm; nitrobenzoxadiazole [NBD]: &#955;ex = 475 nm, &#955;em = 545 nm). 
	NOTE: See Figure 1 for examples of spectrophotometric analyses of bacteria using the fluorescent NBD-labeled ciprofloxacin antibiotic.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amicon Ultra-0.5 centrifugal filter unit with Ultracel- 10 membrane</td>
			<td>Merck</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker Avance 600 MHz spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCCP</td>
			<td>Sigma-Aldrich</td>
			<td>C2759</td>
			<td></td>
		</tr>
		<tr>
			<td>Celite 545</td>
			<td>Sigma-Aldrich</td>
			<td>22140-5KG-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cygel</td>
			<td>ABCAM</td>
			<td>Ab109204</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64FX, fixable analog of FM&#8482; 4-64 membrane stain</td>
			<td>Life Technologies Australia Pt</td>
			<td>F34653</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma 2-16 LSCplus lyophilise</td>
			<td>CHRIST</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hettich Zentrifugen Rotofix 32</td>
			<td>Hettich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB</td>
			<td>AMRESCO</td>
			<td>J106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme from chicken egg white 
			lyophilized powder</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Mueller Hinton II Broth Cation adjusted</td>
			<td>Becton Dickinson</td>
			<td>212322</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma 1-15 Microcentrifuge</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TECAN Infinite M1000 PRO</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells

Source: Stone, M. R. L., et al. <a href="https://app.jove.com/v/60743">Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells. Exposure to an efflux pump inhibitor disrupts the transmembrane proton gradient in bacterial cells, upon which a fluorophore-conjugated antibiotic is added. Inhibition of efflux pumps due to the disrupted proton gradient causes accumulation of the antibiotic inside the bacteria. Bacterial cells are lysed, and the fluorescence intensity is measured to confirm antibiotic accumulation.

Table 1. Antibiotic activities of fluorescent antibiotic probes based on ciprofloxacin, trimethoprim, and linezolid against appropriate clinically relevant bacterial strains, as measured by broth microdilution MIC assays. In most cases, the probes lost some activity compared to the parent drug, but retained some measurable antibiotic potency (sufficient to be useful in further studies).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td c...

An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells

1. Evaluation of Antimicrobial Activity
NOTE: All work involving bacteria should be carried out under sterile conditions to avoid contamination of either ...

Take assay tubes containing bacterial samples.
In the test sample, add inhibitor molecules to block the antibiotic efflux pump of the bacteria.
Centrifuge and discard the supernatant.
Add fluorophore-conjugated antibiotic probes that enter the bacterial cells.
In the control sample, the efflux pump extrudes the probes into the extracellular medium.
In the inhibitor-treated sample, the blocked efflux pump cannot expel the probes, causing their accumulation.
Centrifuge the samples. Discard the supernatant containing extracellular probes.
Add lysis buffer followed by lysozyme to weaken the bacterial cell wall.
Perform freeze-thaw cycles, sonication, and high-temperature exposure to lyse the cells.
Centrifuge. Transfer the supernatant to a filter membrane.
Wash and collect the filtrate containing the probes.
Measure the fluorescence intensity of the filtrate.
A higher fluorescence intensity in inhibitor-treated bacterial samples suggests loss of efflux function, leading to intracellular probe accumulation.

an-assay-to-measure-accumulation-fluorescent-antibiotic-probe

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

56 Views. Source: Stone, M. R. L., et al. Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes. J. Vis. Exp. (2020). This video demonstrates an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells. Exposure to an efflux pump inhibitor disrupts the transmembrane proton gradient in bacterial cells, upon which a fluorophore-conjugated antibiotic is added. Inhibition of efflux pumps due to the disrupted proton gradient causes accumulation of the antibiot...

Video: An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells - Concept

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

JoVE Journal

Biochemistry

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Neuroscience

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells

Transcript

An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells