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1. Viability quantification of GC aggregations
<ol>
	<li>Collect GC using a sterile applicator. Swab GC from the plate and re-suspend GC in pre-warmed broth(GCP, Table 1) supplemented with 4.2% NaHCO3 and 1% Kellogg solutions. Use spectrophotometry at a wavelength of 650 nm (an OD 650 of 1 = ~1 x 109 CFU/mL) to determine the concentration of suspended bacteria.</li>
	<li>Adjust the concentration of GC to ~1 x 108 CFU/mL.</li>
	<li>Add 99 &#181;L of adjusted GC suspension into wells of a 96-well plate.</li>
	<li>Incubate the plate for 6 h at 37 &#176;C with 5% CO2 to allow the bacteria to aggregate.</li>
	<li>Add 1 &#181;L of serial diluted ceftriaxone (1000, 100, 50, 25, 12.5, 6.2, 3.1, 1.5, 0.8, 0.4, 0.2 &#181;g/mL) into each well. Leave some wells untreated to serve as controls.</li>
	<li>Incubate the plate for 24 h at 37 &#176;C with 5% CO2.</li>
	<li>Sonicate the suspension 3 times in each well for 5 s at 144 W and 20 kHz.</li>
	<li>Add 100 &#181;L of commercially available ATP utilization glow reagent into each well, pipette up-and down for 3 times, and incubate for 15 min at 37 &#176;C with 5% CO2.</li>
	<li>Carefully transfer 150 &#181;L of mixture from each well into a new well in a 96-well black microplate and avoid introducing bubbles.</li>
	<li>Measure the absorbance of each well at 560 nm using the plate reader.</li>
	<li>Calculate the survival rate by the ratio of the reading obtained after serial ceftriaxone treatment to the reading from untreated wells.</li>
</ol>
2. Fluorescence microscopic analysis of Live/Dead of GC aggregates
<ol>
	<li>Collect GC using a sterile applicator. Swab GC from the plate and re-suspend GC in pre-warmed GCP media plus 1% Kellogg supplements.</li>
	<li>Determine the number of bacteria by spectrophotometry at a wavelength of 650 nm and adjust the concentration of GC to ~1 x 107 CFU/mL.</li>
	<li>Add 198 &#181;L of GC suspension into in 8-well coverslip-bottom chambers.</li>
	<li>Incubate the chamber for 6 h at 37 &#176;C with 5% CO2 to allow aggregation formation.</li>
	<li>Add 2 &#181;L of ceftriaxone (100 &#181;g/mL or various dilutions) into each well within each aggregation condition. Incubate for the desired time at 37 &#176;C with 5% CO2.</li>
	<li>Add 0.6 &#181;L of live/dead staining solution mixture into each well and incubate for 20 min at 37 &#176;C with 5% CO2.</li>
	<li>Acquire Z-series images using a confocal microscope (an equivalent microscope can be used).</li>
	<li>Analyze the images using ImageJ software for measurement of the size of GC aggregates and the fluorescence intensity ratio FIR of live-to-dead staining in each aggregate.</li>
</ol>
Table 1: Recipe for 1 L of GCP Bacterial Growth Media.
<img alt="Equation1" src="/files/ftp_upload/22010/22010_Table1.jpg" />

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100x Kellogg&#39;s supplement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>United States Biological</td>
			<td>A0930</td>
			<td></td>
		</tr>
		<tr>
			<td>BacTiter Assay</td>
			<td>Promega</td>
			<td>G8232</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceftriaxone</td>
			<td>TCI</td>
			<td>C2226</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco GC medium base</td>
			<td>BD</td>
			<td>228950</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferric nitrate, nonahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>254223-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP350-1</td>
			<td></td>
		</tr>
		<tr>
			<td>BacLight live/dead staining</td>
			<td>Invitrogen</td>
			<td>L7012</td>
			<td></td>
		</tr>
		<tr>
			<td>MS11 Neisseria gonorrhoeae strain</td>
			<td></td>
			<td></td>
			<td>kindly provided by Dr. Herman Schneider, Walter Reed Army Institute for Research</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Kontes</td>
			<td></td>
			<td>Equivelent to 9110001</td>
		</tr>
	</tbody>
</table>

an atp utilization assay for determining bacterial survival under antibiotics treatment

Source: Wang, Liang-Chun, et al. <a href="https://app.jove.com/v/58978">Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining.</a> J. Vis. Exp. (2019).
This video describes the simple ATP-measuring assay using luciferase glow reagent. This assay is used to quantify Neisseria gonorrhoeae survival after treatment with ceftriaxone.

An ATP Utilization Assay for Determining Bacterial Survival Under Antibiotics Treatment

1. Viability quantification of GC aggregations

	Collect GC using a sterile applicator. Swab GC from the plate and re-suspend GC in pre-warmed broth(GCP, ...

Begin by seeding a bacterial culture into an assay plate. Then, incubate the plate.
The bacterial cells have surface adhesins and pili that enable them to adhere to each other, forming small aggregates.
Add varying antibiotic concentrations to the wells, leaving one untreated for a control.
The antibiotic molecules interact with sensitive bacteria, affecting their ability to grow and reproduce, leading to their death.
The antibiotic-resistant bacteria remain metabolically active, producing ATP molecules.
Use a sonicator to disrupt the bacterial cells, releasing their intracellular contents, including ATP.
Add ATP-utilization glow reagent containing luciferin, luciferase enzyme, and other co-factors.
Luciferase utilizes ATP and oxygen to convert luciferin to an excited oxyluciferin.
The oxyluciferin subsequently transitions to its ground state, emitting luminescent light.
Measure the absorbance using a plate reader.
The decrease in absorbance at higher antibiotic concentrations suggests lower ATP production, indicating a reduced bacterial survival rate.

an-atp-utilization-assay-for-determining-bacterial-survival-under

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

96 Views. المصدر: وانغ, ليانغ تشون, وآخرون. الفحص الكمي لقابلية المضادات الحيوية لمجاميع النيسرية البنية باستخدام المقايسات التجارية لاستخدام ATP والتلوين الحي / الميت. J. Vis. Exp. (2019). يصف هذا الفيديو مقايسة قياس ATP البسيطة باستخدام كاشف توهج لوسيفيراز. يستخدم هذا الاختبار لتحديد بقاء الن...

Video: اختبار استخدام ATP لتحديد بقاء البكتيريا تحت العلاج بالمضادات الحيوية - Concept

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

عالية الإنتاجية في المختبر المقايسة باستخدام الجهازيات الورم المستمدة من المريض

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

JoVE Journal

Cancer Research

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

فحوصات قابلية للخلايا في الثقافة

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Biology

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A large share of ATP is needed to re-install ion gradients across plasma membranes degraded by electrical signaling of neurons. There is evidence that astrocytes - while not generating fast electrical signals themselves - undergo increased production of ATP in response to neuronal activity and support active neurons by providing energy metabolites to them. The recent development of genetically encoded sensors for different metabolites now enables the study of such metabolic interactions between neurons and astrocytes. Here, we describe a protocol for cell-type specific expression of the ATP-sensitive Fluorescence Resonance Energy Transfer- (FRET-) sensor ATeam1.03YEMK in organotypic tissue slice cultures of the mouse hippocampus and cortex using adeno-associated viral vectors (AAV). Furthermore, we demonstrate how this sensor can be employed for dynamic measurement of changes in cellular ATP levels in neurons and astrocytes upon increases in extracellular potassium and following induction of chemical ischemia (i.e., an inhibition of cellular energy metabolism).

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

We describe a protocol for cell-type specific expression of the genetically encoded FRET-based sensor ATeam1.03YEMK in organotypic slice cultures of the mouse forebrain. Furthermore, we show how to use this sensor for dynamic imaging of cellular ATP levels in neurons and astrocytes.

التصوير من ATP داخل الخلايا في شرائح الانسجه العضوية من الدماغ الماوس باستخدام الاستشعار القائم علي التاكل ATeam 1.03يمك

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

An ATP Utilization Assay for Determining Bacterial Survival Under Antibiotics Treatment

Transcript

An ATP Utilization Assay for Determining Bacterial Survival Under Antibiotics Treatment