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 — 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.
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