Measuring Phagocytosis using pHrodo Bioparticles

Materials

• pHrodo BioParticles Conjugates (Invitrogen cat #A10010)
• HBSS
• OptiMEM

Protocol

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