Frustrated Phagocytosis

Before the Experiment

1. Prepare substrates and place them into the wells of a 12-well plate. These can be prepared by coating slides with a ligand of interest, or by microprinting.
2. Transfect cells if required, as per usual protocols.
3. Split macrophages into wells without coverslips, 1 well/frustrated phagocytosis assay (~250,000 cells/well).

Experiment: Fixed-Cell

1. Pre-warm the substrates and centrifuges to 37C.
2. If needed, pre-label or treat cells with inhibitors.
3. Wash the cells 3× with warmed PBS.
4. Add ~300 μl Accuatse to each well of cells, incubate at 37C until the cells begin to lift (~10 min).
5. Gently scrape cells, then add an equal volume of medium with FBS to inactive the Accuatase.
6. Pellet cells with a 300×g, 3 min centrifugation.
7. Remove supernatant, being careful to not disturb the pellet.
8. Resuspend cells in 500 μl/well of serum-free medium.
9. Transfer cells onto pre-warmed substrates, then centrifuge using a plate spinner for 1 min at 1,000 × g.
10. Incubate for desired period of time at 37°C/5% CO₂.
11. Fix cells with 4% PFA in PBS, 20 min, using PFA pre-warmed to 37°C.
12. Label cells as required for the experiment.

Experiment: Live-Cell

1. Setup microscope and pre-heat the stage-top incubator, substrates, and Leiden chamber.
2. If needed, pre-label or treat cells with inhibitors.
3. Recover the cells from a single well, using steps 3-8 of the fixed cell protocol.
4. Place a substrate in the Leiden chamber, load with cells, and transfer to the microscope.
5. Focus on the substrate and select several positions to image.
6. Perform time-lapse/point visiting acquisition.
7. Repeat steps 2-6 for the remaining samples.