Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens

Published: November 30, 2023

Abstract

Source: Paschall, A. V. et al., Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens. J. Vis. Exp. (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.

Protocol

1. Culture, Differentiation, and Validation of HL-60 Cells 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. For propagation/maintenance of HL-60 cells, culture 5 x 106 cells in 10 mL of HL-60 cell culture media in 75 cm2 vented flasks at 37 °C and 5% carbon diox…

Representative Results

Figure 1: Validation of HL-60 cell differentiation via flow cytometry. Differentiated HL-60 cells were harvested, washed, and resuspended in 1 x 105 cells/mL PBS. Cells were then aliquoted into 12 wells (100 µL/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…

Divulgaciones

The authors have nothing to disclose.

Materials

Annexin V (APC conjugated) BioLegend 640919
Anti-CD35, human (PE conjugated) BioLegend 333405
Anti-CD71, human (PE conjugated) BioLegend 334105
Bacterial strain to be used (ie, Streptococcus pneumoniae, WU2) Bacterial Respiratory Reference Laboratory (Dr. Moon Nahm) 
Blood agar plates Hardy Diagnostic A10
Fetal Clone serum HyClone SH30080.03
Glycerol Sigma G9012-1L
HL-60 cells ATCC CCL-240
IgG Isotype Control (PE conjugated) BioLegend 400907
N,N-dimethylformamide (DMF) Fisher Chemical UN2265
Propidium iodide Sigma P4864
RPMI media with L-glutamine Corning 10-040-CV

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Citar este artículo
Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens. J. Vis. Exp. (Pending Publication), e21798, doi: (2023).

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