Using Image-Based Flow Cytometry to Assess Monocyte Oxidized LDL Phagocytosis Capacity

Fig. 1

Monocyte and phagocytosis gating. Using all collected events, single cells are identified using a dot plot of bright-field aspect ratio vs. bright-field area (a) followed by the identification of monocytes using a dot plot of CD14 vs. SSC (b) After the monocyte population is identified, a daughter plot is generated of oxLDL vs. CD14 for the “inhibitor” (c) and respective “non-inhibitor” (d) files. The “inhibitor” treated sample is used to set the position of the oxLDL-population. By applying this position to the “non-inhibitor” sample it is possible to identify CD14⁺/oxLDL⁺ events

Create a daughter plot from single cells of CD14 vs. side scatter (SSC) to positively identify monocytes (CD14⁺) (Fig 1b).

Create a daughter plot from CD14⁺ cells of oxLDL vs. CD14. Identify the negative population in the inhibitor treatment sample (Fig 1c) and set a gate to the right of this population to identify monocytes (CD14⁺) that have ingested oxLDL in the non-inhibitor sample (Fig 1d).

Save the file as a template and use the batch-processing wizard to apply the template and compensation matrix to the .rif files for all samples (see Note 15 ).

4 Notes

Stock solutions should be prepared in a biological safety cabinet to maintain sterility.

All fluorescent antibodies were titrated in a series of experiments completed prior to this method with the exception of the oxLDL. A final concentration of 15 μg/mL for oxLDL is recommended by the manufacturer.

Unconjugated CD16 was purchased from eBioscience and conjugated in our laboratory using a PE/Atto594 lighting-link kit from Innova Biosciences (Cambridge, UK). PE/Atto594 has a similar emission profile as PE/Texas Red.

Create an antibody cocktail by adding equal parts of each antibody to a light resistant tube. This will reduce variability by limiting the number of pipette transfers.

Steps 3–5 of Subheading 3.1 should be completed in a Class 2 Biological Safety cabinet or similar equipment to maintain assay sterility.

Plate map example can be seen in Fig. 2.

Fig. 2

Plate map example. Patient samples should be separated by treatment condition (inhibitor or no inhibitor) using rows and columns respectively. Organization of any other condition depends on the assay

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