Frustrated Phagocytosis

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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.