Neutrophil Phagocytosis Assay

Materials

  • Fluorescently labeled bacteria, yeast, microspheres, or other test organism
  • HBSS with calcium and magnesium, but no phenol red (Invitrogen 14025)
  • HBSS, no calcium, magnesium, or phenol red (Invitrogen 14175)
  • Human AB serum, optional opsonizing reagent (MediaTech 35-060-CI)
  • Human neutrophils (Astarte Biologics)
  • 4% paraformaldehyde (CytofixTM; BD 554655)
  • Non-pyrogenic, sterile, low-retention 2.0-mL microtubes 
A.     Preparation of bacteria
  1. Fluorescently label bacteria, yeast or other test organism in a manner appropriate for the study. E. coli, S. aureus, or zymosan BioParticles® from Invitrogen are commonly used by many groups. Fluorescent microspheres can also be used.
  2. Opsonize bacteria or microspheres if desired. Bacteria can be opsonized by incubation in 20% human AB serum (v/v) at a concentration of 109 cells/mL.  Incubate at 37 C for 1 hour.
  3. Dilute bacteria or zymosan samples in HBSS (with Ca/Mg) to give a 10-fold higher concentration than desired in the phagocytosis assay.  To achieve a bacteria:phagocyte ratio of 10 in the below phagocytosis assay, dilute the opsonized bacteria 1:10 to a yield a final concentration of 108 bacteria/mL.
B.     Preparation of human neutrophils
  1. Thaw vial of primary human neutrophils quickly in a 37°C water bath. Slowly add 1 mL of cells to 9 mL of room-temperature HBSS (with calcium/magnesium). Centrifuge at 400 x g for 10 min to pellet cells and gently resuspend in 5 mL of room-temp HBSS.
  2. Count cells with hemacytometer and resuspend at a concentration of 106 cells/mL in room-temp HBSS.
  3. Pipet 0.5 mL of cell suspension into each of desired number of 2.0mL tubes. Transfer tubes to a 37°C incubator for 10 min to warm prior to continuing with the phagocytosis assay.
C.     Opsonophagocytosis assay
  1. Add 50 μL of bacterial or microsphere suspension from step A3 to each microtube containing neutrophils. Add 50 μL of HBSS to at least one tube to create a negative (i.e., no bacteria) control for flow cytometry gating.
  2. Mix solutions very gently by inverting tubes several times. Place tubes in an incubated oven and rotate very gently (~5-10 rpm) for 10 min.
  3. Remove tubes from incubator and immediately place on ice to arrest the phagocytosis process. Immediately add 0.55 mL of cold 4% paraformaldehyde to each tube, mix gently by inverting tubes, and incubate on ice for 15-30 min.
  4. Rinse cells once with cold HBSS (no Ca/Mg) by centrifugation at 400 x g for 10 min. Resuspend cells in 0.2 mL of cold HBSS (no Ca/Mg). 
  5. Measure cell-associated fluorescence by flow cytometry.

This procedure was kindly provided by Kristy Katzenmeyer, Ph.D. of the University of Washington.