Frustrated Phagocytosis
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Before the Experiment
- 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.
- Transfect cells if required, as per usual protocols.
- Split macrophages into wells without coverslips, 1 well/frustrated phagocytosis assay (~250,000 cells/well).
Experiment: Fixed-Cell
- Pre-warm the substrates and centrifuges to 37C.
- If needed, pre-label or treat cells with inhibitors.
- Wash the cells 3× with warmed PBS.
- Add ~300 μl Accuatse to each well of cells, incubate at 37C until the cells begin to lift (~10 min).
- Gently scrape cells, then add an equal volume of medium with FBS to inactive the Accuatase.
- Pellet cells with a 300×g, 3 min centrifugation.
- Remove supernatant, being careful to not disturb the pellet.
- Resuspend cells in 500 μl/well of serum-free medium.
- Transfer cells onto pre-warmed substrates, then centrifuge using a plate spinner for 1 min at 1,000 × g.
- Incubate for desired period of time at 37°C/5% CO2.
- Fix cells with 4% PFA in PBS, 20 min, using PFA pre-warmed to 37°C.
- Label cells as required for the experiment.
Experiment: Live-Cell
- Setup microscope and pre-heat the stage-top incubator, substrates, and Leiden chamber.
- If needed, pre-label or treat cells with inhibitors.
- Recover the cells from a single well, using steps 3-8 of the fixed cell protocol.
- Place a substrate in the Leiden chamber, load with cells, and transfer to the microscope.
- Focus on the substrate and select several positions to image.
- Perform time-lapse/point visiting acquisition.
- Repeat steps 2-6 for the remaining samples.