Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

Summary

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Abstract

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

Introduction

The opsonophagocytic killing assay (OPKA) is a critical tool for linking alterations in bacterial structure or function to subsequent changes in immune response and function. As such, it is frequently used as a complementary assay to determine immune-based efficacy of antibody treatments, vaccine candidates, enzyme optimization, etc. While in vivo assays are necessary to determine effective clearance or protection in a bacterial infection model, the OPKA can be used to assess immune contribution to bacterial cell death at the most basic components: bacteria, immune cells, and experimental treatments. Previous studies have shown that OPKAs can be modified and used for ....

Protocol

1. Culture, Differentiation, and Validation of HL-60 Cells

1. Prepare HL-60 cell culture media composed of 500 mL 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.
2. 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% CO₂. Passage cells every 3−4 days to maintain optimal cell concentrations.
   NOTE: The cell concentration should not exceed 5 x 10⁶/mL.
3. Generate working s....

Results

Validation of HL-60 differentiation should be performed before starting the OPKA. This can be accomplished using flow cytometry to determine the extracellular expression of CD11b, CD35, CD71, and annexin V (Figure 1). Propidium iodide can also be used as a viability marker. After being treated with DMF for 3 days, expression of CD35 should be increased (≥55% of all cells) and expression of CD71 should be decreased (≤20% of all cells). The percentage of annexin V+ and propidium io.......

Discussion

OPKAs serve essential roles in assessing antibody mediated immune responses induced by vaccinations^6,8. The main significance of this simplified OPKA is the adaptability in the conditions to be tested (i.e., antibodies, enzyme treatments, etc.). In this sense, while this assay can be used to test the contribution of opsonins (i.e., antibodies) in phagocytosis, it can also be used to assess ways to overcome virulence factors (i.e., capsular polysaccharides) that n.......

Materials

Name	Company	Catalog Number	Comments
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