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Briefly, TIFFs were seeded at a density of 50,000 cells per ml in a 24-well plate.
Fresh TRIzol® was used to make up volume to 1.5 mL in a 2 mL microtube, before adding 0.3 mL chloroform.
After cooling, the solution was filtered and diluted to 25 mL in a volumetric flask.
Benzoic acid (1 mmol) was dissolved in pyridine (10 mL) in a screw capped vial.
Dilution of 0.1 mL was performed with ethanol up to 10 mL in a volumetric flask.
The organic mixture was emulsified by adding to water (50 ml) in a drop-wise manner.
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Cells were inoculated at 0.5 × 106 cells/ml in a volume of 150 ml in 500 ml shake flasks.
The final protein concentration was determined to 19.6 mg/ml in a volume of 0.7 ml.
Harvest cells, wash twice with 1 ml PBS and suspended in PBS (10x10/ml) in a glass tube.
The suspension was diluted to a start concentration of 2.1×107 bacteria/ml in a final volume of 10 ml.
Patients received infiltration with 300 mg ropivacaine (0.2%) and epinephrine (10 mg/mL) in a total volume of 150 mL.
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