Rhodamine phalloidin reagent

For immunostaining, membrane sheets were fixed at room temperature (RT) for 30 min in 4% paraformaldehyde (PFA) in PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄, pH 7.4) followed by PFA quenching for 20 min with 50 mM NH₄Cl in PBS. Then, membrane sheets were permeabilized with 0.2% Triton X-100 in PBS for 2 min, followed by blocking with 4% bovine serum albumin (BSA; catalog no.: P06-1391100) in PBS for 1 h at RT. Afterward, cover slips were incubated with primary antibody diluted in 1% BSA–PBS for 1 h at RT, followed by three washing steps with 0.5% BSA–PBS, and incubation with secondary antibody diluted in 1% BSA–PBS. Finally, samples were washed three times in PBS. For confocal and STED microscopy, for detection of the membranes from nonoverexpressing SH-SY5Y cells, Vybrant DiO Cell-Labeling Solution (catalog no.: V22886; Thermo Fisher Scientific) in a dilution of 1:200 in PBS or in overexpressing HepG2 cells, Rhodamine Phalloidin Reagent (catalog no.: ab235138; Abcam) in a dilution of 1:1000 was added, and cover slips were mounted on microscopy slides using ProLong Gold antifade mounting medium (catalog no.: P36930; Invitrogen).

The polylysine-based crosslinkers, PLL and PLLGA, were synthesized by ring opening reaction of their corresponding N-carboxyanhydrides [45 (link)]. CNF was synthesized as previously described [75 (link)]. Chemicals for the characterization of the hydrogel structure are ruthenium tetroxide, 0.5% stabilized aqueous solution RuO₄(aq), (20427-56-9, Polysciences Inc., Warrington, DC, USA), and Au nanoparticles (AC11-500-CIT-DIH-100-1-EP, Nanopartz, Loveland, CO, USA). Reagents for in vitro experiments are as follows: AlamarBlue™ cell viability reagent (DAL1025, Thermofisher, Waltham, MA, USA), nerve growth factor-2.5S from the murine submaxillary gland (NGF, N6009-4X25UG, Sigma, St. Louis, MI, USA), rhodamine phalloidin reagent (AB125138, Abcam, Cambridge, MA, USA), 4′,6-diamidino-2-phenylindole (DAPI, D1847, Sigma-Aldrich, St. Louis, MI, USA), Hank’s balanced salt solution, (1×), with calcium, magnesium, without phenol red (HBSS, SH30588, HyClone, Logan, UT, USA), Fluo-2 AM, green fluorescent Ca²⁺ binding dye (ab142775, Abcam, Cambridge, MA, USA), adenosine triphosphate (ATP, A2383, Sigma-Aldrich, St. Louis, MI, USA), Dulbecco’s modified eagle medium (DMEM/F12 1:1, SH30023.02, HyClone, Logan, UT, USA), and RPMI 1640 with L-glutamine (SH30011.02, HyClone, Logan, UT, USA).

For the analysis of immune synapse formation, activated lymphocytes pre-stained with ViaFluor^® 405 (1:1000 dilution, Biotium, Fremont, CA, USA, 30068) and hTERT-HME1 pre-stained with CellTrace™ CFSE Cell (1:1000 dilution, ThermoFisher, C34554) were co-cultured at a 1:1 ratio for 2 h. Cell conjugates were then transferred to poly-L-lysin-coated coverslips. The interaction between lymphocytes and hTERT-HME1 was determined by staining the actin cytoskeleton polymerization with Rhodamine Phalloidin Reagent (1:1000 dilution, Abcam, ab235138), which highlights the F-actin at the immune synapse level. Images were acquired with a Zeiss Axio Vert. A1 inverted microscope, equipped with an Axiocam Camera 503 Mono and analyzed with ZEN Software (Carl Zeiss, Jena, Germany).
The immune synapse formation was quantified as previously reported [31 (link)]. Briefly, three different fields of each sample were analyzed, and the number of lymphocytes linked to hTERT-HME1 were quantified; the percentage of synapse formation was calculated as follows: $$\% of synapse formation= \frac{number of synapses}{numer of synapses +number of lymphocytes alone} \times 100$$

The aBMSCs of the CCD patient, control cells, and transfected HEK293 cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific). Non-specific binding was blocked with 1% bovine serum albumin. The cells were then incubated with the rabbit monoclonal anti-RUNX2 antibody (1:500, Abcam, Cambridge, UK, Cat. No. ab192256), followed by the secondary antibody, the donkey anti-rabbit IgG Alexa Fluor® 488-tagged (1:1000, Biolegend®, Cat. No. 406416), in combination with DAPI (1:2000, Roche, Cat. No. 10236276001) and Rhodamine Phalloidin Reagent (1:500, Abcam, Cat. No. ab235138). The cells were visualized using ApoTome.2 (Zeiss GmbH, Jena, Germany) at 100X magnification.