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.
1. Culture, Differentiation, and Validation of HL-60 Cells
2. Preparation of OPKA Buffers and Reagents
3. Preparation of Bacterial Stock Samples
4. Bacterial Treatment and Culture
5. HL-60 bacterial Co-culture
6. Sample Plating and Overnight Incubation
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 stained with fluorescently conjugated isotype antibodies were used as controls.
Figure 2: Treatment Y improves HL-60-mediated cell killing of bacteria. S. pneumoniae samples were treated with treatment X (antibody) or treatment Y (enzyme). OPKA was performed according to the protocol and bacterial CFUs were counted in duplicate. Samples that were not treated with HL-60 cells were used as a control (100% cell survival). Shown are the average percentages of bacterial CFUs in the HL-60-treated groups compared to the corresponding non-HL-60-treated groups. Bars represent standard error.
The authors have nothing to disclose.
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 |