Name: real-time quantification of reactive oxygen species in neutrophils infected with meningitic escherichia coli
Uploaded: 2021-04-20T13:30:00
Description: Watch this Scientific Journal Video about Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli at JoVE.com

This work was supported by the National Natural Science Foundation of China (31670845, 31870832, 32000811) and the Program of Distinguished Professor of Liaoning Province (LJH2018-35).

Peripheral blood from volunteers applied in this research was approved by the Institutional Review Board of the first Hospital of China Medical University (#2020-2020-237-2).
1. Preparation of reagents and culture medium
<ol>
	<li>Prepare the red blood cell lysis buffer by adding 8.29 g of NH4Cl, 1 g of KHCO3, 37.2 mg of Na2EDTA into 1 L of double distilled water and adjust the pH to 7.2-7.4. Remove the bacteria by filtration using 0.22 &#181;m filters.</li>
...

<ol>
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Neutrophils act as the most abundant component of white blood cells in human blood circulation. They are important effector cells in the innate human immune system, which builds the first line of defense against the invasion of pathogens11. The generation of ROS represents one of the major bactericidal mechanisms of neutrophils following phagocytosis11. Recent studies have shown that a net-like structure released by a neutrophil called neutrophil extracellular trap (NET) is...

Neonatal bacterial meningitis is a common pediatric infectious disease. Escherichia coli (E. coli) with a K1 capsule is the most common Gram-negative pathogen causing neonatal bacterial meningitis, accounting for about 80% of the total incidence1,2,3. Despite the advances in the antimicrobial chemotherapy and supportive care, bacterial meningitis is still one of the most devastating conditions with high morbidity and mortality4.
The occurrence of neonatal bacterial meningitis usually begins with bacteremia caused by the entry of pathogenic bacteria into the peripheral circulation from the local lesions of the newborns, followed by penetration through the blood-brain barrier (BBB) into the brain, resulting in the inflammation of the meninges4. The onset of bacteremia depends on the interaction between bacteria and host immune cells including neutrophils and macrophages, etc. Neutrophils, which account for ~50-70% of white blood cells, are the first line of defense against bacterial infections5,6. During the invasion of bacteria, the activated neutrophils are recruited to the infectious sites and release reactive oxygen species (ROS) including the superoxide anion, hydrogen peroxide, hydroxyl radicals, and singlet oxygen7. The ROS undergo redox reactions with the cell membrane, nucleic acid molecules and proteins of the bacteria, resulting in the injury and death of the invading bacteria8. The mitochondria is the main site of ROS production in eukaryotic cells, and various oxidases (e.g., nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, lipoxygenase system, protein kinase C and cyclooxygenase system) mediate the production of ROS9,10. The real-time measurement of the production of ROS, representing the primary antimicrobial mechanism in neutrophils, is a useful method for studying host defense during the bacteria-host interaction.
In this protocol, the time-dependent ROS production in neutrophils infected with meningitic&#160;E. coli was quantified with a fluorescent ROS probe DHE, detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in other mammalian cells during the pathogen-host interaction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL polypropylene conical centrifuge tubes</td>
			<td>KIRGEN</td>
			<td>KG2611</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>3025</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>DINGGUO</td>
			<td>DH010-1.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Autuomated cell counter</td>
			<td>Bio-rad</td>
			<td>508BR03397</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Safety Carbinet</td>
			<td>Shanghai Lishen</td>
			<td>Hfsafe-1200Lcb2</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion</td>
			<td>BD</td>
			<td>237500</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16 Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-045-701</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Changsha Xiangyi</td>
			<td>TDZ5-WS</td>
			<td></td>
		</tr>
		<tr>
			<td>Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihydroethidium (DHE)</td>
			<td>MedChemExpress</td>
			<td>104821-25-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Cellmax</td>
			<td>SA211.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Heraeus</td>
			<td>Hera Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separation buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M5</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Beyoitme</td>
			<td>S1819-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS separation Unit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Solarbio</td>
			<td>13292-46-1</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 medium</td>
			<td>Sangon Biotech</td>
			<td>E600027-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic shaker</td>
			<td>Shanghai Zhicheng</td>
			<td>ZWY-100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypton</td>
			<td>OXOID</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time quantification of reactive oxygen species in neutrophils infected with meningitic escherichia coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Using the protocol outlined in this article, the neutrophils were isolated from human peripheral blood and loaded with fluorescence probe DHE to detect the changes of ROS levels in response to E44 infection. Here, we provide representative data demonstrating the ROS production evoked by E44 strain determined by a microplate reader in real-time. By adding E44 strains at a MOI of 100, the ROS levels increased immediately and showed a continuous upward trend with a time-dependent manner (Figure 1</stron...

Watch this Scientific Journal Video about Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli at JoVE.com

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...

Escherichia Coli is the most common gram-negative bacteria causing neonatal meningitis. The occurrence of neonatal bacterial meningitis purely begins with the bacteremia and then the bacteria in the blood penetrate through the blood brain barrier to cause meningitis. Neutrophils are the first line of defense against the bacterial infections.During bacterial invasion, the active neutrophils release ROS. ROS represents the major bacteriocidal mechanisms of host cells to destroy the invading pathogens. Measuring the production of ROS in neutrophils is a useful method for starting host's defense during bacteria-host interaction.Take a single bacterial colony from the plate with a sterile pipette and put it in five milliliter Brain-Heart infusion medium containing Rifampicin in a concave flask. Incubate the bacterial culture at 37 centigrade, with 90 RPM for 17 hours in the incubation shaker. Centrifuge the peripheral blood samples at 2000 RPM for five minutes.After centrifuge, the blood samples are divided into three layers. From the bottom to the top, the red blood cells layer, the white blood cells layer, and the plasma layer. Aspirate the white blood cells layer to a new tube with a sterile pipette.Add threefold red blood cell lysis buffer. Blend the mixture thoroughly, and place at room temperature for five minutes. Centrifuge the tube at 2000 RPM for five minutes.Aspirate the supernatant completely and discard. Repeat the lysis procedure of red blood cells for one or two times, until the sediments turn white. Wash the cells by re-suspending the sediment with two milliliter PBS.Then centrifuge the tube at 1000 RPM for five minutes. Resuspend the sediment with 15 microliter precode magnetic cell sorting buffer for 15 million cells. Add 15 microliter magnetic microbeads and mix thoroughly.Incubate the mixture at four centigrade for 30 minutes. Under normal conditions, the adult human being have about four to 10, 000 white blood cells per microliter blood. So the cost of total white blood cells in five milliliter human peripheral blood, nearly less than 50 million.And 50 microliter magnetic beads in 15 microliter magnetic sorting buffer is enough. The normal percentage of the neutrophils is 50 to 70%of the total white blood cells. So about 10 million neutrophils can be obtained from five milliliter blood.Wash the cells by adding two milliliter magnetic sorting buffer, per 10 million cells to the tube, and centrifuge at four centigrade, 1, 200 RPM for 10 minutes. Assemble the magnetic column and separating shelf. Discard the supernatant completely, and resuspend the sediments with 500 microliter magnetic sorting buffer for up to 100 million cells.Rinse the column with three milliliter magnetic sorting buffer. Apply the cell suspension onto the column to allow the neutrophils labeled by the magnetic beads, attached to the column. Washed off the non-specific specific cells by adding three milliliter buffer for three times, once the column reservoir is empty each time.Remove the column from the magnetic separator, and put it in a new tube. Add five milliliter magnetic sorting buffer into the column. Push out the magnetic labeled cells using a plunger.Centrifuge the tube at 1, 200 RPM for five minutes. Aspirate the supernatant completely, and resuspend the sediment with one milliliter culture medium. Determine the cell number with a counter.Adjusted the cell concentration to 2 million per milliliter in the culture medium containing DHE fluorescence probe. Place the neutrophils in the incubator for 30 minutes to load the DHE probe. Allocate the cell suspension to a 96-well black microplate with 200 microliter per well.Turn on the microplate reader. Set the temperature to 37 centigrade, choose fluorescence read mode, kinetic read type, set the fluorescence wavelength. Choose the plate format.Determine the reading area. Measure the fluorescence intensity every five minutes for an hour. Shake the plate for three seconds before each reading.Take out the microplate from the incubator. Add PMA or E44 strains with three repetitions. Put the plate in the microplate reader, press the start button to begin the assay immediately.Using the protocol outline in this article, the neutrophils were isolated from the human peripheral blood, and loaded with fluorescence probe DHE. By adding PMA, intracellular ROS in neutrophils, showed a significant increase from 20 to 40 minutes, and reached peak at 60 minutes. But I think E44 strains, the ROS production occurs immediately and increases time dependently.The expression of ROS caused by E44 strain is similar to that caused by PMA. The detection of ROS production in neutrophils may contribute to the further study of the bacteriocidal mechanisms of neutrophils. In this protocol, we provide an easier way to detect the ROS production in neutrophils in a real-time manner.The simple method could also be used to monitor the ROS production in other hosts cell types during host-bacteria interactions.

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