Annexin V-FITC Apoptosis Detection Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Prepare 1x Binding Buffer by diluting 1 ml of the 10x Binding Buffer with 9 ml of deionized water. - Dissolve the staurosporine in DMSO to a concentration of 100 mg/ml. - cells were pretreated with BER for 1 h, then cultured with or without fMLP (1µM) stimulation for 12 h		- Apoptotic neutrophils are here defined as Annexin V-positive but propidium iodide-negative cells.

Upstream tips
- Prepare 1x Binding Buffer by diluting 1 ml of the 10x Binding Buffer with 9 ml of deionized water. - Dissolve the staurosporine in DMSO to a concentration of 100 mg/ml. - cells were pretreated with BER for 1 h, then cultured with or without fMLP (1µM) stimulation for 12 h
Downstream tips
- Apoptotic neutrophils are here defined as Annexin V-positive but propidium iodide-negative cells.

Publication protocol

To measure the cytotoxicity of BER on neutrophils, isolated neutrophils were seeded in 24-well plates, which cells were pretreated with BER for 1 h, then cultured with or without fMLP (1µM) stimulation for 12 h. Stimulated cells were next collected, stained with FITC-conjugated (fluorescein isothiocyanate) Annexin V and propidium iodide following the manufacturer’s instruction, then analyzed on a FACS-Canto II(BD Biosciences). Apoptotic neutrophils are here defined as Annexin V-positive but propidium iodide-negative cells.

To determine the effects of BER on activation of neutrophils, isolated neutrophils were seeded in 24 well-plates, then pretreated with BER for 1 h before stimulation with fMLP (1µM) for 2 h. Thus-prepared cells were collected, suspended in staining buffer (1 % BSA in PBS), then stained with FITC-conjugated anti-CD11b and PE-conjugated anti-CD62L at 4 °C for 30 min. Finally, the conjugated samples were washed with staining buffer and analyzed by flow cytometry.

From inoculated mice, peritoneal lavage fluids were concentrated, suspended in staining buffer and stained with APC-conjugated anti-Gr-1, FITC-conjugated anti-CD11b and PE-conjugated anti-F4/80 at 4 °C for 30 min. Then, the samples were washed and analyzed by flow cytometry. Identification of macrophages was based on positive labeling for F4/80 and CD11b, while neutrophils were identified by positive labeling for Gr-1 and CD11b.

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