Admin: Created page with "==== 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. # I..."

2024-07-10T15:12:48Z

Created page with "==== 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. # I..."

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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 [[Micropatterning Proteins|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% CO<sub>2</sub>.
# Fix cells with 4% PFA in PBS, 20 min, using PFA pre-warmed to 37°C.
# [[Immunostaining|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.

Frustrated Phagocytosis - Revision history