A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester

Abstract

Apoptotic cell phagocytosis has recently raised considerable interest, particularly due to its intricate molecular mechanisms and negative immunologic impact of incompetent clearance of apoptotic cells. There is a need for simple and reliable methods to clearly determine the internalization of apoptotic cells. Labeling with pHrodo succinimidyl ester (SE), a pH-sensitive fluorescent dye, makes engulfed apoptotic cells detectable due to the increased post-phagocytic light emission. This is a valuable tool for phagocytosis studies via FACS. We designed an ex vivo assay, using apoptotic pHrodo-labeled lymphocytes as prey and anti-CD11b-labeled tissue macrophages. To demonstrate its validity of detecting internalized apoptotic lymphocytes, we used MFGE8 -/-macrophages, known to have impaired phagocytic ability. Uptake of apoptotic lymphocytes was accelerated and enhanced in splenic macrophages after stimulation with recombinant MFGE8, while peritoneal macrophages were able to compensate for the delayed uptake. This novel assay is a quick and reliable method to evaluate the internalization of apoptotic cells.

Nucleolin Is a Receptor for Maleylated-Bovine Serum Albumin on Macrophages

“…4,8,21) Macrophages were suspended at 5×10 5 cells/mL in RPMI-1640 medium buffered with 20 mM 4-[2-hydroxyethy]1-piperazineethanesulfonic acid (RPMI 1640-HEPES, pH 7.2) and then incubated with 0-20 µg/ mL Alexa Fluor ® 488-labeled control-BSA or maleylated-BSA at 37°C for 2 h. After 2 h, the cells were incubated with phycoerythrin-labeled anti-CD11b antibody for 30 min on ice, then washed and immediately analyzed using a flow cytometer (FACS Calibur, Becton-Dickinson, Franklin Lakes, NJ, U.S.A.) with CellQuest software (Becton-Dickinson) and gating for forward scatter (FSC) and side scatter (SSC) regions of intact cells. Since fluorescent labeled anti-CD11b was used for selection of macrophages at flow cytometer analysis, antiCD11b positive cells were regarded as macrophages.…”

“…Since fluorescent labeled anti-CD11b was used for selection of macrophages at flow cytometer analysis, antiCD11b positive cells were regarded as macrophages. 4,8,21) Confocal Images of Phagocytosed Maleylated-BSA Macrophages were seeded on a coverslip as previously described 8) and incubated with 0-10 µg/mL Alexa Fluor ® 488-labeled control-BSA or maleylated-BSA, and membranes were stained with the fluorescent cell-linker compound PKH 26 red. BSAs taken up by macrophages were identified using confocal laser-scanning fluorescence microscopy (FV1000D; Olympus, Tokyo, Japan).…”

Phagocytosis of neonatal pathogens by peripheral blood neutrophils and monocytes from newborn preterm and term infants

“…pHrodo is a highly pH-sensitive succinimidyl ester dye that fluoresces dramatically when exposed to low pH (<4.0). Phagocytosis is therefore detectable and quantifiable when the phagosome forms, and acidifies, around ingested pHrodo-labeled bacteria, minimizing high background fluorescence of cell-bound, but uningested, bacteria (18,19).…”

Nucleolin Acts as a Scavenger Receptor for Acetylated Low-Density Lipoprotein on Macrophages

“…Thioglycollate-elicited murine macrophages were isolated from male ddY mice (Japan SLC, Inc., Shizuoka, Japan), as previously described. 10,12,20) Macrophages were suspended at 5×10 5 cells/mL in RPMI-1640 medium buffered with 20 mM HEPES (pH 7.2) and then incubated with 0 to 10 µg/mL DiI-labeled normal LDL or AcLDL in the presence or absence of anti-nucleolin antibody, control rabbit IgG, AGRO or CRO at 37°C for 2 h. After 2 h, cells were incubated with Alexa Fluor 488-labeled anti-CD11b antibody for a further 30 min on ice and then washed. Cells were immediately analyzed using a flow cytometer (FACS Calibur; Becton Dickinson, Franklin Lakes, NJ, U.S.A.) with CELLQUEST software (Becton Dickinson) and gating for forward scatter (FSC) and side scatter (SSC) regions of intact cells.…”

“…CD11b-positive cells were regarded as macrophages. 10,12,20) Binding of Normal LDL and AcLDL to NucleolinTransfected HEK Cells Human nucleolin cDNA was transfected into HEK 293 cells (HEK cells; Health Science Research Bank, Osaka, Japan) as previously described. 10) After transfection, adherent HEK cells were detached from the bottom of the culture well by incubation with Puck's EDTA solution (5 mM HEPES, 0.1 M NaCl, 5 mM potassium chloride (KCl), 4 mM sodium bicarbonate (NaHCO 3 ), 1 mM EDTA, and 5.6 mM glucose) for 10 min and gentle pipetting.…”