Tube revolver

SF microparticles were prepared by the atomization method, in which SF aqueous solution was sprayed into an absolute ethanol coagulation bath. The atomization system consists of a nozzle with a diameter of 0.5 mm (Labmaq, Ribeirão Preto, São Paulo, Brazil), a peristaltic pump TE-BP-01 Mini (Tecnal, Piracicaba, São Paulo, Brazil), and a compressor model OP8.1/30II (Pressure, Maringá, Paraná, Brazil). The SF solution was pumped (flow rate around 0.045 L/h) to the atomizing nozzle, where the solution was broken up into smaller droplets (compressed air pressure at 1 bar). The droplets came into contact with the absolute ethanol under stirring. After forming the microparticles, they were kept at rest for 30 min and then stirred for 10 min.
Each dye solution (1 × 10⁻⁴ mol/L) was incorporated into the microparticles by adsorption, by immersing 0.1 g of SF microparticles into 10 mL of dye solution and stirring at 30 rpm by a tube revolver (Thermo Scientific, Waltham, MA, USA) until the equilibrium (3 h) at room temperature (24 °C). The dyes used were MB, RB, RhB, and NR. These dyes were selected due to their different properties showed in Table 1. The rational selection was made to verify the influence of the charge, molar mass, and hydrophilicity of the dye in the SF matrices. MB, RB, and RhB are anionic dyes, and NR is a slightly cationic dye at neutral pH.

(Knockout) cells were grown in a 6-well plate until confluent, washed twice with cold PBS, and lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with phosphatase inhibitors (PhosSTOP EASYpack; Roche) and protease inhibitors (EASYpack protease inhibitor cocktail; Roche). After 5 min of incubation at 4°C, cells were scraped, transferred into Eppendorf tubes, and incubated in a tube revolver (Thermo Fisher Scientific) for 1 h at 4°C. Samples were centrifuged for 15 min at 15,000 × g at 4°C. The supernatants were diluted in NuPAGE LDS sample buffer (Supplier: Thermo Fisher Scientific) (4×) and analyzed by Western blot analysis as described previously (45 (link)). Monoclonal antibodies targeting RIG-I (AG-20B-0009-C100; Bio-Connect), PKR (ab226819; Abcam), and MAVS (ab89825; Abcam) were used according to the manufacturer’s instructions. Secondary polyclonal rabbit anti-mouse IgG-horseradish peroxidase (HRP) (P0260; Agilent) and polyclonal swine anti-rabbit IgG-HRP (P0217; Agilent) antibodies were used at a 1:1,000 dilution. Imaging was performed using an Amersham imager 600 (GE Healthcare).

Different RBC:NP ratios (1:50, 1:100, 1:500 and 1:1000) were tested. Relevant ratios were chosen based on previous works [15 (link), 16 (link)]. Ratios were calculated based on nanoparticle concentration and RBC count. Equal volumes (150 ul) of RBC solution and NP suspension were mixed in Axygen 1.5-ml Self-Standing Screw Cap Tubes mixed by pipetting and inversion. The tubes were then rotated on a tube revolver (Thermo Fisher Scientific) at 12 rpm for 30 min. The coupled RBCs were then pelleted by centrifugation at 100 g for 5 min at 4 °C and the pellet was washed again with 1 ml of cold PBS to remove loosely bound NPs. The supernatant was analyzed for hemoglobin presence (absorbance read at 550 nm) with a plate reader (SpectraMax iD3, Molecular Devices), to quantify RBC hemolysis during coupling. The pelleted, coupled RBCs were finally resuspended at 10% hematocrit in PBS.

Resected tumors were transported in Hank’s Balanced Salt Solution (HBSS, Life Technologies) on ice immediately after surgery. The tumor sample was subsequently divided into two pieces, and a small fragment was stored in liquid nitrogen for tissue staining. The remainder of the tumor was minced with scalpels into tiny cubes <0.5 mm³ and transferred into a 15 mL conical tube (BD Falcon) containing 8 mL pre-warmed HBSS, 1 mg/mL collagenase I and 0.5 mg/mL collagenase IV. Tumor pieces were digested on a Tube Revolver (Thermo) for 30 min at 37 °C. This suspension was then filtered using a 70 μm nylon mesh (BD Biosciences) and residual cell clumps were discarded, then the cell pellet was resuspended in red blood cell lysis buffer. Following a 5 min incubation at room temperature, samples were centrifuged to discard the supernatant and re-suspend the cell pellet in PBS with 0.04% FBS. Cell sorting was performed with a MoFloAstrios EQ (Beckman Coulter). Live cells were used for single-cell experiments after the dead cells were eliminated based on exclusion of 7-aminoactinomycin D (Life Technologies).