Alexa fluor 594 conjugated phalloidin

Planar bilayers were formed by using small liposome droplets with clean glass coverslips as described previously. Briefly, biotin-labeled CD5 and CD7 proteins (ACRO system) were added to the bilayer and incubated for 30 min at 37 °C. After they were washed, 1.0 × 10⁵ CAR⁺ T cells of each group were suspended in T-cell medium and incubated with the lipid bilayers for 15 min at 37 °C. Then, the cells were fixed for 30 min with 4% paraformaldehyde at 4 °C. To visualize the immune synapse, F-actin was stained with Alexa Fluor 594-conjugated phalloidin (Abcam) following the manufacturer’s instructions. Subsequently, a FITC-conjugated AffiniPure F(ab)'2 fragment-specific goat anti-mouse IgG antibody (Jackson ImmunoResearch) or FITC-conjugated anti-gamma tubulin antibody (Abcam) was used to stain CARs or MTOCs, respectively. Nuclei were stained with Hoechst (Sigma) for 5 min. Confocal images were acquired by a Leica SP8 confocal laser-scanning microscope. All images were confirmed not to be overexposed by the software. All images were processed and analyzed with LAS X (Leica) and ImageJ (National Institutes of Health).

The PA copolymers with compositions of 300PEOT55PBT45 (PA 300) and 1000PEOT70PBT30 (PA 1000), where 300 and 1000 are the molecular weight in g mol⁻¹ of the starting polyethylene glycol blocks used during the copolymerization and the ratios of the PEOT:PBT segments are 55:45 and 70:30, respectively, were purchased from PolyVation BV. Chloroform, 1,1,1,3,3,3-Hexafluoro-2-propanol (HFiP), fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA), phosphatase buffered saline (PBS), Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (pen-strep), formalin, triton-X solution, and DAPI were purchased from Sigma-Aldrich. The Alexa Fluor 594 conjugated phalloidin was purchased from ABCAM. Rat Schwann cells (rSCs), cell line RT4-D6P2T, were purchased from ATCC.