Acti stain 488

5 × 10⁵ T cells in RPMI/0.5% BSA were incubated with 100 ng/mLSDF-1α, fixed with 4% cold formaldehyde, permeabilized (0.1% Triton X-100), and F-actin was stained with Actistain 488 (Cytoskeleton, Denver, CO, USA) for 1 h on ice, followed by flow cytometry.

C2C12 myoblasts were cultured as described previously (Pęziński et al., 2019 (link)). Briefly, myoblasts were cultured on 0.2% gelatin-coated plates in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 4.5 g/L glucose, L-glutamine, penicillin, streptomycin, and fungizone. For myotube fusion, cells were trypsinized and plated on Permanox slides (VWR) in eight-well Flexiperm chambers (Sarstedt). The slides were coated with 111-laminin (Sigma). To induce myotube differentiation, the media were replaced with DMEM with 2% horse serum. For experiments involving agrin, slides were coated with 0.2% gelatin instead of laminin, and myotubes were stimulated with 10 nM recombinant rat Z’-spliced agrin (R&D Systems) for 72 h. The cells were fixed in 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100 for 30 min. The myotubes were stained using Alexa Fluor 555-coupled bugarotoxin (BTX) (B35451, Life Technologies) and Acti-Stain 488 (PHDG1, Cytoskeleton). Cells were imaged on the Zeiss spinning disk confocal microscope at the Nencki Institute Microscopy Core Facility. Images were processed using ImageJ/Fiji and analyzed in R.