Le 80k

Polysome fractionation protocol was adapted from Stastna et al (Stastna et al, 2018 ). Briefly, Fmr1^+/+ and Fmr1^−/− BMDM were treated with PA (500 µM) for 6 h followed by cycloheximide (100 µg/ml) for 10 min prior to lysis with buffer (100 mM KCl, 20 mM Tris pH 7.5, 5 mM MgCl₂, 0.4% NP‐40, 100 µg/ml cycloheximide, 0.1 U RNase inhibitor and protease inhibitor cocktail). Clear lysates were loaded to 10–50% sucrose gradient (in Beckman Coulter Thinwall, Ultra‐Clear tubes, 344059) and centrifuged (in Beckman LE‐80K) for 120 min at 28,4061 g at 4°C in a swinging bucket rotor (Beckman SW41) with no‐brake. Each gradient was collected as 17 fractions in microcentrifuge tubes with continuous monitoring of absorbance at 254 nm (Biologic LP (pump), Biorad 731‐8300; BioFrac, Biorad 741‐0002) and frozen immediately at −80°C for further analysis.

The photos from the colony formation assays and animal study were collected by Nikon D3200 (pixel dimension: 3020*1728, 8 bit) from October, 2011 to December, 2014 in Tianjin Life Science Research Center. EGFP and RFP intensity was tested using an F-4500 fluorescence spectrophotometer (HITACHI, Tokyo, Japan). EGFP intensity is normalized to RFP intensity. qRT-PCR was performed using the Bio-RAD™ iQ5 fluorescence quantitative PCR instrument. Lab Works™ Image Acquisition and Analysis Software (UVP) were used for the quantification of the western blot bands in the form of gray intensities (pixel dimension: 3020*1728, 8 bit, UVP, U.S.A.) from October, 2011 to January, 2015 in Tianjin Life Science Research Center. The Sucrose gradient sedimentation assay were performed with the Beckman ultracentrifugation LE-80 K with SW28ti rotor at 25,000 rpm for 4 hrs at 4 °C.

To acquire the cell membranes for nanoparticle coating, T cells and CAR-T cells were washed by PBS twice and then harvested. The cells were suspended in hypotonic lysing buffer consisting of 20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl₂, and 1 EDTA-free mini protease inhibitor tablet per 10 mL of solution and disrupted using a dounce homogenizer with a tightfitting pestle. The entire solution was subjected to 20 passes before spinning down at 3,200 g for 5 min. The supernatant was saved, while the pellet was resuspended in hypotonic lysing buffer and subjected to another 20 passes and spun down again. The supernatants were pooled and centrifuged at 20,000 g for 30 min, after which the pellet was discarded and the supernatant was centrifuged again at 80,000 g for 1.5 h using an ultra-speed centrifuge (LE-80K, Beckman Coulter, USA). The pellet containing the plasma membrane material was then washed once with 10 mM Tris-HCl and 1 mM EDTA and collected. Then, CAR-T vesicles (CVs) and T cell vesicles (TVs) were obtained by physically extruding the pellet for 11 passes through a 400-nm polycarbonate porous membrane on a mini extruder (Avanti Polar Lipids, USA).