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  • Fluorescently Labeled Bacteria, Yeast, Microspheres, or Other Test Organism
  • HBSS with Calcium and Magnesium, But No Phenol Red (Invitrogen 14025)
  • HBSS with No Calcium, Magnesium, or Phenol Red (Invitrogen 14175)
  • Human AB Serum, Optional Opsonizing Reagent (MediaTech 35-060-CI)
  • Human Neutrophils
  • 4% paraformaldehyde (CytofixTM; BD 554655)
  • Non-pyrogenic, Sterile, Low-retention 2.0-mL Microtubes

This method has been used to measure phagocytosis by neutrophils from Cellero.

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 109cells/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.

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 minutes to pellet cells and gently resuspend in 5 mL of room temperature HBSS.
  2. Count cells with hemacytometer and resuspend at a concentration of 106 cells/mL in room temperature HBSS.
  3. Pipet 0.5 mL of cell suspension into each of desired number of 2.0 mL tubes. Transfer tubes to a 37°C incubator for 10 minutes to warm prior to continuing with the phagocytosis assay.

Opsonophagocytosis Assay

  1. Add 50 μL of bacterial or microsphere suspension from Preparation of Human Neutrophils Step 3 (above) 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 minutes.
  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 minutes.
  4. Rinse cells once with cold HBSS (no Ca/Mg) by centrifugation at 400 x g for 10 minutes. 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.