Sigma 4k15 centrifuge

Liposomes composed of DOPE, CHEMS e DSPE-PEG₂₀₀₀ and DSPE-PEG₂₀₀₀-DTPA (SpHL-DTPA-PTX) at a molar ratio of 5.7:3.8:0.45:0.05, respectively, were prepared using the standard lipid film hydration method [3 (link)]. In brief, pre-determined chloroform aliquots of the lipids and PTX (0.5 mg/ml) were transferred to round bottom flask and a lipid film was obtained by evaporating the organic solvent under reduced pressure. Next, to promote the complete ionization of CHEMS molecules, an aliquot of NaOH solution (0.456 M) was added at a 1:1 molar ratio CHEMS:NaOH. The film was hydrated with NaCl 0.9% (w/v), followed by vigorous shaking in vortex. The vesicles were sonicated (20% amplitude) in an ice bath for 5 min using a high-intensity ultrasonic processor (R2D091109 model; Unique^® Instruments, Indaiatuba, Brazil). The suspension were submitted to a centrifugation process (Sigma 4k-15 centrifuge, Sigma Laborzentrifugen GmbH, Osterode, Germany) at 3000 rpm at 4 °C for 10 min to eliminate non-entrapped PTX. Since the drug shown low water solubility, it precipitate and a drug pellet is formed. The supernatant suspension represent the final and purified formulation. For the folate-coated liposomes, 0.05% of DSPE-PEG₂₀₀₀-folate was added to the lipid film formation.

B. fragilis cultures (20 mL) were harvested at 3,000 × g for 10 min at 4°C in a Sigma 4K15 centrifuge. The pellet was re-suspended and washed in 1 × phosphate-buffered saline (PBS) and re-pelleted again at 3,000 × g for 10 min at 4°C. Afterwards bacteria were re-suspended in a small volume (400 µL) of ultrapure water and proteins were precipitated by adding 12.5% trichloroacetic acid (TCA) in acetone to the sample until a final concentration of 10% TCA was reached. Proteins were precipitated for at least 1 h at -20°C. The precipitates were pelleted in a Sigma 1-15PK cryo-centrifuge (20,000 × g, 20 min, 4°C) and pellets were washed in 90% acetone (20,000 × g, 20 min, 4°C). Afterwards pellets were dried and re-suspended in 2DE sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 1% ampholytes (pH 3-10) for at least 3 h. Insoluble matter was removed by centrifugation at 20,000 × g for 20 min at 20°C. The protein concentration in the supernatant was determined by Bradford assay (Bradford, 1978 ) and 400 µg of protein were used for 2DE.