Figure 1.

Before you begin preparation of materials

(A) Image depicting pHrodo-labeled protein-opsonized beads (pink arrowhead) settled in the ACSF (yellow arrowhead) in a 1.5 mL microcentrifuge tube, either after the centrifuge step during washing or after long term storage at 4 °C.

Note the pink coloration of the settled beads, marking the pHrodo-red label’s presence.

(B) Image depicting the resuspended bead mixture after vortexing or rapid pipetting (blue arrowhead). Note the milky white coloring, as the pHrodo-red label is diluted in the ACSF.

(C–F) Depictions of the tissue containers used for slice incubation and drug incubation of slices.

These containers have two spaces - one part to hold the slices containing a mesh to allow ACSF exchange and one part that houses the source of the CO2 gas exchange in the ACSF.

Purple arrowheads indicate where the mesh that the slices will sit on is present. This is either nylon stretched over the cups in the general slice storage or the bottom of the 40 μm filters used in the drug incubation storage.

Green arrowheads indicate the space outside of the filtered area to accommodate the bubbler (as gas should not be bubbled directly onto the slices to preserve slice health).

Red arrowheads indicate the gas line running the CO₂ mix into the ASCF, to continue to preserve slice health. The orange arrowhead and inset in E demonstrates where the 40 μm filter’s edge has been cut so that it better fits into the 20 mL beaker.

Seen in markings along the filter rim near the orange arrowhead is a label denoting which drug incubation this filter is associated with.

As demonstrated in F, separate system set ups are needed per drug and vehicle incubation.