Beckman l8 70m ultracentrifuge

Pig blood was obtained under Wisconsin State Inspection in the UW-Madison Meat Science and Muscle Biology Laboratory. Four volumes of pig blood were mixed thoroughly with 1 vol of anticoagulant containing 150 mM NaCl and sodium heparin (120 Units/ml). To remove the plasma, washing buffer (4 volumes of 1.7 % NaCl in 1 mM Tris, pH 8.0) were added to heparinized blood and centrifuged (700 g for 10 min at 4 °C) in a Beckman J-6B centrifuge (Beckman Instruments Inc., Palo Alto, CA). The red blood cells were washed three times more using 10 volumes of the same washing buffer (Fyhn, Fyhn, Davis, Powers, Fink, & Garlick, 1979 ). After which, 3 volumes of stock solution (1 mM Tris, pH 8.0) were added to lyse cells for 30 min. One-tenth volume of 1 M NaCl was then added to aid in stromal removal, and it was centrifuged (28,000 g for 15 min at 4 °C) in a Beckman L8-70 M ultracentrifuge (Beckman Instruments Inc., Palo Alto, CA). Hemolysates were then stored at −80 °C until use.

ABA tri-block copolymer, PEG5K-b-oligo(desaminotyrosyl-tyrosine octyl ester suberate)-b-PEG5K (Fig. 1A), was synthesized, characterized, and prepared using previously established protocols21 (link). Briefly, TyroSpheres loaded with and without CSA (referred to as CSA-TyroSpheres or empty, respectively) were prepared as follows. 20% (w/v) polymer (e.g., 60 mg/0.3 mL) and 10, 20, 30, 40 and 60 wt.% CSA (e.g., 20 mg/mL, equivalent to 10 wt.% initial loading) were dissolved in DMF, mixed in equal amounts (0.3 mL each) and added drop-wise to 1xPBS (14.4 mL) under constant stirring. The resulting suspension was passed through 0.22 μm PVDF filters (Millipore) and ultracentrifuged for 3 h at 18 °C and 65,000 rotations per minute (rpm) (290 000 × g ) (Beckman L8-70M ultracentrifuge, Beckman Coulter, Fullerton, CA). The supernatant was aspirated and the CSA-TyroSpheres were rinsed twice with 1 mL 1xPBS. Then, the CSA-TyroSpheres were re-suspended in 1 mL 1xPBS, ultracentrifuged under the same conditions, and re-suspended in 1 mL 1xPBS (pH 7.4) overnight at 25 ± 2 °C.

MSCs were transfected for 48h, starved overnight (o.n.), and grown in starvation medium for 72h if not indicated differently. Breast cancer or epithelial cells were grown for 72h in complete growth medium. Subsequently, CM was retrieved, cell debris was removed by centrifugation and stored immediately at -80°C and thawed only once for experimental usage. For ultracentrifugation (UC), the CM was first centrifuged for 30min at 2,000g to remove dead cells and cell debris. The residual supernatant was centrifuged at 100,000g for 180min using a SW 41 Ti Rotor in a Beckman L8-70M Ultracentrifuge (Beckman Coulter, California, USA). Separation of proteins larger than 4kDa in CM of cancer cells was performed with AMICON® Ultra-4 filtration units (Merck Millipore, Darmstadt, Germany) at 4000g for 30 minutes.
MSC CM was analyzed for secreted proteins using Human Cytokine Array Kit, Panel A (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to manufacturer's instructions except that IRDye®800CM Streptavidin (LI-COR, Lincoln, NE, USA) was used in a 1:4000 dilution in PBS for visualization. Membranes were scanned with Odyssey Reader and analyzed with Odyssey 2.1 (LI-COR, Lincoln, NE, USA). Fluorescence linked-immunosorbent assay (FLISA) was used for protein quantification in CM (Protocol in Supplemental Materials).

Ten sucrose solutions from 50% to 5% (w/v) sucrose were prepared in 10 mM Tris (pH 7.5), 1 mM EDTA (pH 8) and 100 mM NaCl as previously described^{20 (link)}. First, 1 mL of the 50% sucrose solution was added to the bottom of the tube (Beckman Coulter, 344059) and flash frozen in liquid nitrogen. Then each sucrose solution was layered on top of the previous solution and frozen prior to addition of the next layer with the 5% sucrose solution on top of the tube. The sucrose density gradients were stored at −20 °C and thawed slowly on ice before adding protein lysates.
The Control and RNase samples were carefully overlaid on top of the thawed sucrose gradients avoiding any disturbance. For each condition and replicate, 2 to 2.5 mg of proteins were loaded onto the sucrose gradients. Ultracentrifugation was performed in Beckman L8-70M Ultracentrifuge equipped with a SW 41 Ti Swinging-Bucket Rotor (Beckman Coulter, 331362) at 110,000 x g for 18 h at 4 °C. After centrifugation, 25 fractions (~440 μL each) were carefully transferred by pipetting into fresh 1.5 mL tubes. Fraction 1 corresponded to the top of the tube and fraction 25 to the bottom. The different fractions were stored at −80 °C for western blot analysis or precipitated with 20% trichloroacetic acid (TCA) for mass spectrometry.

The 293 cells were grown in DMEM media supplemented with 10% FBS at 37 °C. The designated plasmids are transfected using Lipofectamin 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The supernatant is collected 36 h post transfection. The cells in the supernatant were separated through centrifugation (F33V, from Champion, Gwinnett, GA, USA) at 5000 rpm for 15 min. The supernatant was filtered using 0.4 um filters. The VLPs were segregated from the supernatant by centrifugation at 10,000 rpm for 2 h in Beckman L8-70M Ultracentrifuge and SW41 rotor (Beckman Coulter, Brea, CA, USA) at 4 °C through 1 mL of 10% sucrose. The VLP pellet was resuspended in PBS and stored at 4 °C.