CD11b Antibody, anti-human/mouse, FITC

Protocol tips

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	An aliquot (50 μL) of each was mixed with 50 μL of ice-cold PBS containing FITC-conjugated anti-human CD11b antibody (4 μL, undiluted) and incubated (4°C, 30 min, in darkness). After adding 2 mL of FACS lysing solution (BD Biosciences, San Jose, CA), incubation (room temp, 10 min), tubes were centrifuged (300 x g, room temp, 5 min). The cell pellets were re-suspended in 200μL of PBS and stored in 4 °C in the dark until evaluation by flow cytometry.

Protocol tips

An aliquot (50 μL) of each was mixed with 50 μL of ice-cold PBS containing FITC-conjugated anti-human CD11b antibody (4 μL, undiluted) and incubated (4°C, 30 min, in darkness). After adding 2 mL of FACS lysing solution (BD Biosciences, San Jose, CA), incubation (room temp, 10 min), tubes were centrifuged (300 x g, room temp, 5 min). The cell pellets were re-suspended in 200μL of PBS and stored in 4 °C in the dark until evaluation by flow cytometry.

Publication protocol

Evaluation of neutrophils by flow cytometry was performed as previously described (Uchida et al., 2007). Briefly, 200 μL of whole blood in a 1.5 ml polypropylene tube was incubated (37°C, 30 minutes) with recombinant human GM-CSF (10 ng/mL) or other cytokines / chemokines, placed on ice immediately. An aliquot (50 μL) of each was mixed with 50 μL of ice-cold PBS containing FITC-conjugated anti-human CD11b antibody (4 μL, undiluted) and PE-conjugated anti-human CD16 antibody (4 μL, undiluted) in a polystyrene round-bottom tube (BD Bioscience, San Jose, CA) and incubated (4°C, 30 min, in darkness). After adding 2 mL of FACS lysing solution (BD Biosciences, San Jose, CA), incubation (room temp, 10 min), tubes were centrifuged (300 x g, room temp, 5 min). The cell pellets were re-suspended in 200μL of PBS and stored in 4 °C in the dark until evaluation by flow cytometry. The amount of cell-surface CD11b on neutrophils ([CD11bSurface]) was measured by flow cytometry as mean fluorescent intensity of FITC on CD16Hi cells defined by gating. In some experiments, cytoplasmic CD11b was stained with PE-CD11b (4 μl, undiluted) using the Intracellular Cytokine Staining Kit (eBioscience, San Diego, CA) as per manufacturer's instructions.

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