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…
Divulgations
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 |
Citer Cet Article
Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens. J. Vis. Exp. (Pending Publication), e21798, doi: (2023).