Measuring Phagocytosis using pHrodo Bioparticles

Materials

Subculture cells 3-4 days in advance
On the day of the assay, harvest cells and spin down. Resuspend in OptiMEM at 1 000 000 cells/mL
Plate into a 96 well plate at 100 000 cells per well (100uL of a 1 000 000 cells/mL suspension), plating each condition in triplicate. Leave at least set of wells empty as a blank.
Add 100 uL of OptiMEM to blank wells.
Incubate at least 1h at 37C in a CO2 incubator.
Treat wells as desired.
Thaw one vial of particles for every 20 wells (including no cell controls). Add 2 mL HBSS to a vial and vortex briefly.
Transfer to a clean glass tube and sonicate for 5 min to ensure even dispersion.
After cells have adhered, aspirate media.
Replace media with 100 uL of the resuspended bioparticles.
Put in incubator for 2-3h
Scan plate using 550nm excitation and 600nm emission