An Opsono-Adherence Assay for Assessing the Opsonization of Encapsulated Bacteria by Anti-Capsule Antibodies

Wash the inactivated bacterial stock three times in PBS to remove fixative. Add FITC solution at a final concentration of 75 micrograms per milliliter. Slowly, agitate the mixture for 18 to 24 hours at four degrees Celsius. Wash the bacteria as before to remove excess FITC.

After the final wash, resuspend the bacilli in PBS in the same start volume. Aliquot and store bacilli in -20 degrees Celsius until use. Heat-inactivate a small volume of test sera from non-human primates at 56 degrees Celsius for 30 minutes, in a heat block or PCR machine. Thaw and wash FITC-labeled bacteria with PBS two times by pelleting at 15,000 times g for three to five minutes. Resuspend in PBS in the same start volume.

In a 96-well round-bottom plate, serially dilute the test sera in cell medium. In another 96-well round-bottom plate, add 10 microliters of diluted test sera into each well. Then, add 10 microliters of freshly reconstituted baby rabbit complement.

To plate number three, add the appropriate volume of FITC-labeled bacilli for a multiplicity of infection of 20, for example, at a volume containing 50,000 times 20 bacilli for 50,000 cells. Top off each well in plate number three with the appropriate volume of cell medium to a total of 105 microliters. Incubate plate number three at 37 degrees Celsius for 30 minutes in the presence of 5% CO2 with humidity to opsonize bacilli.

Place plate number one which contains adherent cells on a 37 degrees Celsius heat block. Use a multichannel pipetter to remove spent medium from wells and quickly replace with 100 microliters of opsonized bacteria from plate number three ensuring no bubbles are generated in the wells during pipetting. To not disturb the cell monolayer, add volume to the sides of the wells.

Incubate plate number one at 37 degrees Celsius with 5% CO₂ and humidity for 30 minutes for adherence. Wash each well five times with 150 microliters of PBS on top of a 37 degrees Celsius heat block to remove unattached bacilli. Pipette to the side of the wells to ensure that the cells are not accidentally dispersed during washing. Add 150 microliters of 4% paraformaldehyde to each well, and fix the cells for one hour.

After washing the cells twice with PBS, mix two microliters of high-content screening orange cell stain in 10 milliliters of PBS. Then, add 150 microliters of this staining solution to fixed cells, and incubate for 30 minutes at ambient temperature. Place plate number one on the stage and focus on the cells using bright-field or phase contrast.

In the Acquisition tab, select 96-Well format as the plate setting then select Channel settings. Next, select Setting to Tile Mode. Indicate the number of images to be acquired. Change the acquisition mode to black and white or monochrome, and set the binning to at least 2 by 2. Turn on the autofocus or focusing module. Indicate how often the autofocusing function is to be performed.

Next, turn on the z-stack function if available indicate the number of z-stacks that will capture the thickness of the cell monolayer and adherent bacilli. Designate a file folder to save the images to. Now, run the automated imaging program. Perform data collection and analysis, as described in the text protocol.