MultiTox-Fluor Multiplex Cytotoxicity Assay

Protocol tips

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	Add the 2X MultiTox-Fluor Multiplex Cytotoxicity Assay Reagent in an equal volume to all wells. Mix briefly on an orbital shaker,then incubate for at least 30 minutes at 37°C. Do not incubate longer than 3 hours

Protocol tips
Add the 2X MultiTox-Fluor Multiplex Cytotoxicity Assay Reagent in an equal volume to all wells. Mix briefly on an orbital shaker,then incubate for at least 30 minutes at 37°C. Do not incubate longer than 3 hours

Publication protocol

OMVs were labeled and incubated with the 1% (vol/vol) lipophilic fluorophore dialkylcarbocyanine iodide (Dil) (Invitrogen, Grand Island, NY, USA) for 30 min at 37 °C. The OMV suspensions were ultracentrifuged (150,000 × g, 4 °C, 2 h) and washed three times with DPBS. Labeled OMV particles were pelleted in DPBS and determined the protein concentration to 2 mg/ml solutions by BCA protein assay kit (Thermo Pierce, Rockford, IL, USA) for use.

Murine macrophage RAW264.7 cells were maintained at 37 °C with 5% CO2 in DMEM (Gibco BRL, Grand Island, NY, USA) containing 10% FBS (Hyclone, Logan, UT, USA). Cells were plated onto the wells of a 24-well plate (5 × 105 cells per well) covered with glass coverslips (Biokeystone, CA, USA) and cultured for 24 h. After three washed with PBS, labeled OMVs were added into culture medium at final concentration of 2 μg/ml. After culturing for 2 h, cells were washed three times with PBS and then fixed with 4% formaldehyde in PBS for 10 min, and later cells incubated with OMVs were blocked with SEA block buffer (Thermo Pierce, Rockford, IL, USA) for 1 h at room temperature. Nuclei were labeled with 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen, Grand Island, NY, USA) for 30 min in room temperature. Cells were visualized with AMG EVOS digital inverted multi-functional microscope (Advanced Microscopy Group, Bothell, WA, USA) at 100 × magnification.

To investigate the cytotoxicity of OMVs for macrophages, diverse concentrations of OMVs ranging from 3.075 μg/ml to 100 μg/ml were added to RAW264.7 cells plated in 24-well plates (5 × 105 cells/well). After 24 hours, the supernatants from each well were collected and evaluated by a Multitox-Fluor Multiplex Cytotoxicity Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. All experiments were performed in triplicate

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Manufacturer protocol

Download the product protocol from Promega for MultiTox-Fluor Multiplex Cytotoxicity Assay below.

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