Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens

Overview

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

1. 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.
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% carbon dioxide (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 stocks of HL-60 cells by aliquoting approximately 1 x 10⁶ cells/mL in HL60 culture media with 10% dimethyl sulfoxide (DMSO) into 1 mL cryogenic tubes.
   NOTE: Working stocks may be stored at -80 °C. The master stock should be stored at -120 °C.
4. Differentiate HL-60 cells by culturing 1.5 x 10⁷ cells in 15 mL of HL-60 cell culture media with 0.6% N, N-dimethylformamide (DMF) at 37 °C and 5% CO₂ in sterile filter-capped 75 cm² flasks for 3 days prior to opsonophagocytic killing activity (OPKA).
5. Validate that the HL-60 cells have been successfully differentiated and are appropriate for use in the OPKA assay by testing the viability and cell surface markers according to established flow cytometry protocols. After differentiation, harvest HL-60 cells and stain approximately 1 x 10⁴ cells with fluorescently-conjugated antibodies/stains for CD71, CD35, annexin V, and propidium iodide.
   NOTE: Differentiated cells should be ≥65% viable, ≥55% CD35⁺, and ≤20% CD71⁺ as determined through established validation protocols (Figure 1).

2. Preparation of OPKA Buffers and Reagents

1. Prepare 50 mL of sterile opsonization buffer B (OBB) by mixing 42.5 mL of sterile 1x phosphate-buffered saline (PBS) with Ca²⁺/Mg²⁺, 5 mL of heat-inactivated fetal bovine serum, and 2.5 mL of 0.1% sterile gelatin. Store at 4 °C.
2. Obtain baby rabbit complement and store at -80 °C.
3. Obtain or prepare bacterial culture plates (i.e., 15 x 100 mm² 5% sheep's blood agar plates).

3. Preparation of Bacterial Stock Samples

1. Obtain a stock of the bacterial strain(s) to be tested.
   NOTE: For this protocol, serotype 3 Streptococcus pneumoniae (WU2, generously provided by Dr. Moon Nahm) is used.
2. Grow the bacterial strain in an appropriate broth (i.e., Todd-Hewitt broth + 0.5% yeast extract for this WU2 strain) for approximately 2−4 h at 37 °C.
   NOTE: The optical density at 600 nm (OD₆₀₀) of the culture should be between 0.6 and 0.8.
3. Pellet the bacteria by centrifugation at 6,000 x g for 2 min and resuspend the cells in 10−30 mL of 15% glycerol in the appropriate broth. Aliquot the bacterial culture (500 µL per aliquot) into sterile 1.5 mL centrifuge tubes and store at -80 °C.
4. Thaw out one vial of bacterial stock in a 37 °C water bath. Pellet the bacterial cells and resuspend in 500 µL of OBB under sterile conditions.
5. Prepare different dilutions of the bacterial stock in OBB (i.e., 10 µL of no dilution, 10 µL of 1:10, 10 µL of 1:100, etc.). Perform the OPKA assay (sections 4–6, including HL-60/complement co-culture) as described below using various dilutions of the untreated bacterial stock. Culture the plates overnight at 30 °C (no CO₂).
   NOTE: The temperature of 30 °C is specific for WU2 to prevent overgrowth; other strains/serotypes may grow optimally at 37 °C.
6. Count the colonies for each dilution of untreated bacterial stock co-cultured with HL-60 cells and complement. Determine which dilution of bacteria yields the optimal number of countable colonies (approximately 80−120 colony-forming units (CFUs) for untreated bacteria co-cultured with HL-60 cells). Note this dilution for future OPKAs involving this bacterial stock.

4. Bacterial Treatment and Culture

1. Thaw one tube of bacterial stock prepared in step 3.3. Pellet bacteria (6,000 x g for 2 min) and resuspend the cell pellet in OBB at optimal dilution as determined in step 3.6.
2. Pipette 10 µL of resuspended bacterial dilution per well in a round-bottom 96-well cell culture plate.
3. Add 20 µL of appropriate antibody or drug treatment to each experimental well in duplicate.
   NOTE: In this protocol, a serotype-specific antibody generated in mice is added as treatment X, and a glycoside hydrolase enzyme known to degrade the serotype 3 polysaccharide capsule is added as treatment Y (Figure 2). For control wells, use 1x PBS or OBB, depending on the buffer used for treatment wells.
4. Shake the sample plate at approximately 90 rpm for 1 h at room temperature. Adjust these conditions depending on the optimal temperature or shaking conditions of the treatments being tested.

5. HL-60 bacterial Co-culture

1. Prepare HL-60 cells by harvesting the HL-60 differentiated cells that are treated with DMF three days prior (see step 1.4) into 15 mL conical tubes. Pellet the cells (500 x g, 3 min), discard the supernatant, and wash with at least 10 mL of 1x PBS.
2. Pellet the washed cells (500 x g, 3 min), discard the supernatant, and resuspend the cells in OBB (start with 1 mL OBB and adjust for a final concentration of 1 x 10⁷/mL after cell counting).
3. Add baby rabbit complement (sterile, undiluted baby rabbit serum, age 3−4 weeks) at a 1:5 final volume.
   NOTE: The final concentration of the HL-60-complement mixture should be 1 x 10⁷/mL. If testing complement dependency, a second solution containing active HL-60 cells with heat-inactivated complement may be used (complement may be inactivated by incubating in a water bath at >55 °C for at least 30 min).
4. After 1 h bacterial culture is complete (step 4.4), divide each sample (i.e., 10 µL of each 30 µL sample well into two new wells) into duplicate wells for two groups (i.e., use only 20 µL of the original 30 µL co-culture to account for pipetting error): one set will be co-cultured with HL-60-complement and one will include bacteria only. Add 50 µL of the HL-60-complement mixture (from step 5.3) to each experimental set of wells (delegated +HL-60); add 50 µL of OBB alone to the wells of bacteria only (delegated -HL-60).
   NOTE: For this example, approximately 800 bacterial CFUs are used for the initial co-culture with 5 x 10⁵/50 µL HL-60 cells. If this multiplicity of infection is too high or too low as indicated by final colony numbers, adjust the initial bacterial dilution as opposed to the HL-60 cell count.
5. Shake the 96-well plate at 37 °C for 1 h (no CO₂).

6. Sample Plating and Overnight Incubation

1. Dilute each well 1:5 with OBB, so that each sample has a volume of at least 50 µL.
2. Pipette 50 µL of each sample directly onto a designated area of a bacterial culture plate, ensuring adequate spacing between samples. For 15 x 100 mm² round agar plates, pipet approximately 4 samples onto one plate.
3. Cover and allow samples to dry for approximately 15 min at room temperature.
4. Invert plates and culture overnight at 30 °C (no CO₂). Alternatively, culture plates in anaerobic jars to test whether anoxic conditions affect bacterial growth or to control for morphology.
5. After overnight culture, count the colonies in each designated sample area. Analyze data by comparing the number of live cells in each set to the corresponding control and/or samples that do not receive HL-60 cell co-culture (indicative of 100% cell survival, 0% cell killing).

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 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.

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