Measurement of phagocytosis and of the phagosomal environment in polymorphonuclear phagocytes by flow cytometry

Abstract

Phagocytes are the most important early components of the immune response, programmed to recognize, engulf, and destroy immune complexes (formed when antibodies recognize their specific antigens), foreign particles, bacteria, mycobacteria, apoptotic cells, etc. Neutrophils, monocytes, macrophages, and dendritic cells all participate in this process. Flow cytometry permits observation of phagocytes that have responded and, with the appropriate fluorescent probes, of the environment in the phagosome that has enclosed the foreign matter. This unit gives the background and the protocols for performing such studies.

Figure 1

pHrodo™Bioparticles (Invitrogen/Molecular Probes) were labeled according to Protocol 1 with OregonGreen™ SE and AlexaFluor350™ SE. The fluorescence of a 1μg/ml suspension in PBS at various pH was then measured in a Hitachi 4500 spectrofluorimeter (lexc 565, 488 and 357, lem 590, 530 and 450, respectively). The ratio of F590/F450 and F530/F450 was then calculated. The fluorescence of AlexaFluor350™ did not vary detectably over the pH range. Because the ratios were too disparate to permit reasonable comparison, the % of the ratio at each pH was calculated and is plotted here, the ratios at pH7.68 being arbitrarily defined as 100%.

Figure 2

Mycobacterium avium was triple labeled with AlexaFluor350™/pHrodo™/DCF™ according to Protocol 1, opsonized with autologous serum, and added to PMN at t=0 according to Protocol 2. Fluorescence emission was recorded continuously at 450nm (Alexafluor 350), 530 nm (DCF), or 590nm (pHrodo), a shown in the respective panels. As indicated in the figures’ legends, trypan blue (tpb) was added according to Protocol 3 to distinguish between enclosed (non-quenched) and bound but not enclosed (exposed to trypan blue, therefore quenched) organisms either 15 seconds before (15b) or at the indicated times after the Mycobacterium avium suspension (one second after = 1a, 30 seconds after = 30a). The control, i.e., unquenched, fluorescence is indicated as Ma.

Figure 2

Mycobacterium avium was triple labeled with AlexaFluor350™/pHrodo™/DCF™ according to Protocol 1, opsonized with autologous serum, and added to PMN at t=0 according to Protocol 2. Fluorescence emission was recorded continuously at 450nm (Alexafluor 350), 530 nm (DCF), or 590nm (pHrodo), a shown in the respective panels. As indicated in the figures’ legends, trypan blue (tpb) was added according to Protocol 3 to distinguish between enclosed (non-quenched) and bound but not enclosed (exposed to trypan blue, therefore quenched) organisms either 15 seconds before (15b) or at the indicated times after the Mycobacterium avium suspension (one second after = 1a, 30 seconds after = 30a). The control, i.e., unquenched, fluorescence is indicated as Ma.

Figure 2

Mycobacterium avium was triple labeled with AlexaFluor350™/pHrodo™/DCF™ according to Protocol 1, opsonized with autologous serum, and added to PMN at t=0 according to Protocol 2. Fluorescence emission was recorded continuously at 450nm (Alexafluor 350), 530 nm (DCF), or 590nm (pHrodo), a shown in the respective panels. As indicated in the figures’ legends, trypan blue (tpb) was added according to Protocol 3 to distinguish between enclosed (non-quenched) and bound but not enclosed (exposed to trypan blue, therefore quenched) organisms either 15 seconds before (15b) or at the indicated times after the Mycobacterium avium suspension (one second after = 1a, 30 seconds after = 30a). The control, i.e., unquenched, fluorescence is indicated as Ma.