Shreffler:Monocyte Phagoflow

Materials

Equipment

• Microcentrifuge

Reagents

• Sodium hydrogen carbonate 0.1 M buffered at pH 8.5 with NaOH
• Glycine 0.1 M in PBS
• FITC (F7250-100MG; Sigma-Aldrich Inc), aliquotted at 50 mg/ml, 50 µl/aliquot)
• Polybeads Amino 3.0 Micron Microspheres, 5 mL (Cat# 17145; Polysciences, Inc)

Procedure

Before starting procedure, prepare FITC-conjugated polybeads

Monocyte isolation

1. Isolate PBMCs by ficoll spin. Use 30million PBMCs in Stemcell CD14+ selection for monocytes, using cold staining buffer and a cold magnet for isolation. Warm up water bath to 37°C at this time.
2. Should have a yield of at least 1.5 million cells (typically higher, closer to 6 million cells). Set aside 0.5 million macrophages for CD14-FITC staining to determine purity.
3. Keep cells on ice after isolation.

Phagocytosis

1. Resuspend cell pellet in 95 µl of buffer (RPMI + P/S + 10% FCS). Mix by gently pipetting.