eoe sub articles

EoE Sub Articles

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 diox...

assessment of opsonophagocytic killing activity against bacterial pathogens

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...

Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens

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.