assessment of opsonophagocytic killing activity against bacterial pathogens

Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens

Thaw one tube of bacterial stock. Pellet bacteria at 6,000 times g for two minutes, and resuspend the cell pellet in OBB at the optimal dilution obtained in the previous step. Pipette 10 microliters of the resuspended bacterial dilution per well in a round bottom 96-well cell culture plate. Then, add 20 microliters of an appropriate antibody or a drug treatment to each experimental well in duplicate.
For control wells, use 1X PBS or OBB, depending on the buffer used for the treatment wells. Shake the sample plate at approximately 90 RPM at room temperature for one hour.
To harvest the HL60-differentiated cells that are treated with DMF three days prior, pellet the cells at 500 times g for three minutes. Discard the supernatant and wash the pellet with at least 10 milliliters of 1XPBS. Pellet the washed cells at 500 times g for three minutes. Discard the supernatant and resuspend the cells in OBB.
Add sterile undiluted baby rabbit serum at a one to five final volume. After a one-hour bacterial culture, divide each sample into duplicate wells for two groups. Next, add 50 microliters of the HL60 complement mixture to each experimental set of wells and 50 microliters of OBB to the wells of bacteria only. Shake the 96-well plate at 37 degrees Celsius for one hour.
To plate the samples, dilute each well with OBB at a one to five final volume so that each sample has a volume of at least 50 microliters. Next, pipette 50 microliters of each sample directly onto a designated area of a bacterial culture plate, ensuring adequate spacing between samples. Cover, and allow samples to dry for approximately 15 minutes at room temperature.
Invert the plates and culture overnight at 30 degrees Celsius. Alternatively, culture plates in anaerobic jars, to test whether anoxic conditions affect the bacterial growth or to control for morphology. After overnight culture, count the colonies in each designated sample area and proceed to analyzing data by comparing the number of live cells in each set to the corresponding controls.
The most difficult aspects of establishing this technique for the first time will likely be optimizing the starting CFUs of the bacterial stock, as well as ensuring the HL60 cells are effectively differentiated.

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1. Culture, Differentiation, and Validation of HL-60 Cells
<ol>
	<li>Prepare HL-60 cell culture media composed of 500 mL Roswell Park Memorial Institute (RPMI) with L-glutamine and 50 mL heat-inactivated fetal bovine serum. Do not add antibiotics as this may affect the differentiation of the HL-60 cells.</li>
	<li>For propagation/maintenance of HL-60 cells, culture 5 x 106&#160;cells in 10 mL of HL-60 cell culture media in 75 cm2&#160;vented flasks at 37 &#176;C and 5% carbon dioxide (CO2). Passage cells every 3&#8722;4 days to maintain optimal cell concentrations. 
	NOTE:&#160;The cell concentration should not exceed 5 x 106/mL.</li>
	<li>Generate working stocks of HL-60 cells by aliquoting approximately 1 x 106&#160;cells/mL in HL60 culture media with 10% dimethyl sulfoxide (DMSO) into 1 mL cryogenic tubes. 
	NOTE:&#160;Working stocks may be stored at -80 &#176;C. The master stock should be stored at -120 &#176;C.</li>
	<li>Differentiate HL-60 cells by culturing 1.5 x 107&#160;cells in 15 mL of HL-60 cell culture media with 0.6%&#160;N, N-dimethylformamide (DMF) at 37 &#176;C and 5% CO2&#160;in sterile filter-capped 75 cm2&#160;flasks for 3 days prior to opsonophagocytic killing activity (OPKA).</li>
	<li>Validate that the HL-60 cells have been successfully differentiated and are appropriate for use in the OPKA assay by testing the viability and cell surface markers according to established flow cytometry protocols. After differentiation, harvest HL-60 cells and stain approximately 1 x 104&#160;cells with fluorescently-conjugated antibodies/stains for CD71, CD35, annexin V, and propidium iodide. 
	NOTE:&#160;Differentiated cells should be &#8805;65% viable, &#8805;55% CD35+, and &#8804;20% CD71+&#160;as determined through established validation protocols&#160;(Figure 1).</li>
</ol>
2. Preparation of OPKA Buffers and Reagents
<ol>
	<li>Prepare 50 mL of sterile opsonization buffer B (OBB) by mixing 42.5 mL of sterile 1x phosphate-buffered saline (PBS) with Ca2+/Mg2+, 5 mL of heat-inactivated fetal bovine serum, and 2.5 mL of 0.1% sterile gelatin. Store at 4 &#176;C.</li>
	<li>Obtain baby rabbit complement and store at -80 &#176;C.</li>
	<li>Obtain or prepare bacterial culture plates (i.e., 15 x 100 mm2&#160;5% sheep&#39;s blood agar plates).</li>
</ol>
3. Preparation of Bacterial Stock Samples
<ol>
	<li>Obtain a stock of the bacterial strain(s) to be tested. 
	NOTE:&#160;For this protocol, serotype 3&#160;Streptococcus pneumoniae&#160;(WU2, generously provided by Dr. Moon Nahm) is used.</li>
	<li>Grow the bacterial strain in an appropriate broth (i.e., Todd-Hewitt broth + 0.5% yeast extract for this WU2 strain) for approximately 2&#8722;4 h at 37 &#176;C. 
	NOTE:&#160;The optical density at 600 nm (OD600) of the culture should be between 0.6 and 0.8.</li>
	<li>Pellet the bacteria by centrifugation at 6,000 x&#160;g&#160;for 2 min and resuspend the cells in 10&#8722;30 mL of 15% glycerol in the appropriate broth. Aliquot the bacterial culture (500 &#181;L per aliquot) into sterile 1.5 mL centrifuge tubes and store at -80 &#176;C.</li>
	<li>Thaw out one vial of bacterial stock in a 37 &#176;C water bath. Pellet the bacterial cells and resuspend in 500 &#181;L of OBB under sterile conditions.</li>
	<li>Prepare different dilutions of the bacterial stock in OBB (i.e., 10 &#181;L of no dilution, 10 &#181;L of 1:10, 10 &#181;L of 1:100, etc.). Perform the OPKA assay (sections 4&#8211;6, including HL-60/complement co-culture) as described below using various dilutions of the untreated bacterial stock. Culture the plates overnight at 30 &#176;C (no CO2). 
	NOTE:&#160;The temperature of 30 &#176;C is specific for WU2 to prevent overgrowth; other strains/serotypes may grow optimally at 37 &#176;C.</li>
	<li>Count the colonies for each dilution of untreated bacterial stock co-cultured with HL-60 cells and complement. Determine which dilution of bacteria yields the optimal number of countable colonies (approximately 80&#8722;120 colony-forming units (CFUs) for untreated bacteria co-cultured with HL-60 cells). Note this dilution for future OPKAs involving this bacterial stock.</li>
</ol>
4. Bacterial Treatment and Culture
<ol>
	<li>Thaw one tube of bacterial stock prepared in step 3.3. Pellet bacteria (6,000 x&#160;g&#160;for 2 min) and resuspend the cell pellet in OBB at optimal dilution as determined in step 3.6.</li>
	<li>Pipette 10 &#181;L of resuspended bacterial dilution per well in a round-bottom 96-well cell culture plate.</li>
	<li>Add 20 &#181;L of appropriate antibody or drug treatment to each experimental well in duplicate. 
	NOTE:&#160;In this protocol, a serotype-specific antibody generated in mice is added as treatment X, and a glycoside hydrolase enzyme known to degrade the serotype 3 polysaccharide capsule is added as treatment Y (Figure 2). For control wells, use 1x PBS or OBB, depending on the buffer used for treatment wells.</li>
	<li>Shake the sample plate at approximately 90 rpm for 1 h at room temperature. Adjust these conditions depending on the optimal temperature or shaking conditions of the treatments being tested.</li>
</ol>
5. HL-60 bacterial Co-culture
<ol>
	<li>Prepare HL-60 cells by harvesting the HL-60 differentiated cells that are treated with DMF three days prior (see step 1.4) into 15 mL conical tubes. Pellet the cells (500 x&#160;g, 3 min), discard the supernatant, and wash with at least 10 mL of 1x PBS.</li>
	<li>Pellet the washed cells (500 x&#160;g, 3 min), discard the supernatant, and resuspend the cells in OBB (start with 1 mL OBB and adjust for a final concentration of 1 x 107/mL after cell counting).</li>
	<li>Add baby rabbit complement (sterile, undiluted baby rabbit serum, age 3&#8722;4 weeks) at a 1:5 final volume. 
	NOTE:&#160;The final concentration of the HL-60-complement mixture should be 1 x 107/mL. If testing complement dependency, a second solution containing active HL-60 cells with heat-inactivated complement may be used (complement may be inactivated by incubating in a water bath at &#62;55 &#176;C for at least 30 min).</li>
	<li>After 1 h bacterial culture is complete (step 4.4), divide each sample (i.e., 10 &#181;L of each 30 &#181;L sample well into two new wells) into duplicate wells for two groups (i.e., use only 20 &#181;L of the original 30 &#181;L co-culture to account for pipetting error): one set will be co-cultured with HL-60-complement and one will include bacteria only. Add 50 &#181;L of the HL-60-complement mixture (from step 5.3) to each experimental set of wells (delegated +HL-60); add 50 &#181;L of OBB alone to the wells of bacteria only (delegated -HL-60). 
	NOTE:&#160;For this example, approximately 800 bacterial CFUs are used for the initial co-culture with 5 x 105/50 &#181;L HL-60 cells. If this multiplicity of infection is too high or too low as indicated by final colony numbers, adjust the initial bacterial dilution as opposed to the HL-60 cell count.</li>
	<li>Shake the 96-well plate at 37 &#176;C for 1 h (no CO2).</li>
</ol>
6. Sample Plating and Overnight Incubation
<ol>
	<li>Dilute each well 1:5 with OBB, so that each sample has a volume of at least 50 &#181;L.</li>
	<li>Pipette 50 &#181;L of each sample directly onto a designated area of a bacterial culture plate, ensuring adequate spacing between samples. For 15 x 100 mm2&#160;round agar plates, pipet approximately 4 samples onto one plate.</li>
	<li>Cover and allow samples to dry for approximately 15 min at room temperature.</li>
	<li>Invert plates and culture overnight at 30 &#176;C (no CO2). Alternatively, culture plates in anaerobic jars to test whether anoxic conditions affect bacterial growth or to control for morphology.</li>
	<li>After overnight culture, count the colonies in each designated sample area. Analyze data by comparing the number of live cells in each set to the corresponding control and/or samples that do not receive HL-60 cell co-culture (indicative of 100% cell survival, 0% cell killing).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Annexin V (APC conjugated)</td>
			<td>BioLegend</td>
			<td>640919</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD35, human (PE conjugated)</td>
			<td>BioLegend</td>
			<td>333405</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD71, human (PE conjugated)</td>
			<td>BioLegend</td>
			<td>334105</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial strain to be used (ie, Streptococcus pneumoniae, WU2)</td>
			<td>Bacterial Respiratory Reference Laboratory (Dr. Moon Nahm)&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blood agar plates</td>
			<td>Hardy Diagnostic</td>
			<td>A10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Clone serum</td>
			<td>HyClone</td>
			<td>SH30080.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G9012-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>HL-60 cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Isotype Control (PE conjugated)</td>
			<td>BioLegend</td>
			<td>400907</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide (DMF)</td>
			<td>Fisher Chemical</td>
			<td>UN2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI media with L-glutamine</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Paschall, A. V. et al., <a href="https://app.jove.com/v/59400">Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens.</a>&#160;J. Vis. Exp.&#160;(2019)
The video shows an in vitro opsonophagocytic killing assay using a co-culture of human neutrophils with Streptococcus pneumoniae. Antibodies and complement proteins facilitate opsonization of the bacteria, enabling neutrophils to recognize and kill bacteria, showing successful neutrophil opsonophagocytic activity.

<img alt="Figure 1" src="/files/ftp_upload/21798/21798_Figure1.jpg" /> 
Figure 1: Validation of HL-60 cell differentiation via flow cytometry.&#160;Differentiated HL-60 cells were harvested, washed, and resuspended in 1 x 105&#160;cells/mL PBS. Cells were then aliquoted into 12 wells (100 &#181;L/well) in a 96-well plate. Cells were then stained with fluorescently conjugated anti-CD35, anti-CD71, annexin V, and propidium iodide. Unstained cells or cells st...

1. Culture, Differentiation, and Validation of HL-60 Cells

	Prepare HL-60 cell culture media composed of 500 mL Roswell Park Memorial Institute (RPMI) ...

Begin with a multi-well plate containing a Streptococcus pneumoniae suspension.
Introduce bacterial surface antigen-targeting antibodies, that interact with bacterial surface antigens. This coats the bacteria with antibodies, forming opsonized bacteria for immune recognition.
Seed the wells with human peripheral blood lymphocyte-derived neutrophils supplemented with rabbit serum containing complement proteins.
During incubation, the complement proteins such as C3b get deposited on the bacterial surface, leading to additional opsonin coating on the antibody-opsonized bacteria &#8212; enhancing bacterial recognition by neutrophils.
The specific surface receptors on the neutrophils interact with the opsonins on the bacteria, facilitating bacterial engulfment through phagocytosis.
Inside the phagosome, neutrophils degrade the engulfed bacteria, reducing the bacterial count in the buffer.
Post-incubation, spot the diluted co-culture solution onto a blood agar plate to allow individual bacteria to form colonies.
Compare the number of colonies in opsonin-treated bacteria to untreated bacteria.
Fewer colonies with opsonin treatment indicate higher bacterial killing, suggesting successful opsonophagocytic killing.

60 Aufrufe. Bewertung der opsonophagozytären Abtötungsaktivität gegen bakterielle Krankheitserreger

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Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Alveolären Makrophagen Phagozytose und Bakterien-Clearance bei Mäusen

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

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JoVE Journal

Immunologie und Infektion

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

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Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to&#160;opsono-phagocytic killing assays. An advantage of the opsono-adherence&#160;assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.&#160;&#160;&#160;&#160;&#160;&#160;

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

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Opsono-Adherence Assay zur Bewertung funktioneller Antikörper in der Impfstoffentwicklung gegen Bacillus anthracis und andere verkapselte Pathogene

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Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens

Transkript

Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens