Phalloidin 488

M1 and M2 marker expression in microglia was examined using immunocytochemistry (ICC) according to previously published methods [15 (link)]. Fixed cells were probed with primary antibodies for the M1 phenotype, iNOS (1:200; BD Transduction Laboratories; Franklin Lakes, NJ), and COX-2 (1:200; Thermo Scientific, Rockford, IL) or the M2 phenotype, Arg-1 (1:200; GeneTex, Irvine, CA), RELMα/Fizz1 (1:100; PeproTech, Rocky Hill, NJ), or transglutaminase-2 (1:100; Thermo Fischer Scientific). In addition, the microglial cell markers anti-iba1 (1:5,000; Wako Pure Chemical Industries, Ltd., Tokyo, Japan) and CD68/ED1 (Macrosialin; 1:200; AbD Serotec) were used. For visualization, the secondary antibodies, goat anti-mouse Alexa-594 or anti-rabbit Alexa-594 (1:200, Life Technologies Corp.) were used along with the nuclear marker Hoechst 33342 (1:1,000; Life Technologies Corp.). The morphology of the cells was demarcated by staining with Phalloidin-488 (1:100; Life Technologies Corp). Images were acquired by sequential scanning of the immunostained cells with an Olympus Fluorescence confocal microscope (Olympus, Fluoview FV 1000). For each treatment condition, three to four randomly selected fields were imaged per well. Images obtained were representative of three independently conducted experiments.

Cellular morphology was visualized on Day 2 using fluorescence microscopy. Briefly, samples were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 15 min at room temperature (RT). After rinsing with PBS three times, the samples were placed in a permeabilization solution with 0.1% (v/v) Triton X-100 for 10 min and rinsed again with fresh PBS three times. The cells were incubated with Phalloidin 488 and DAPI (Life Technologies, Carlsbad, CA, USA) to visualize the f-actin and nuclei, respectively.

The assessment of cell alignment was conducted on Day 31 of C2C12 cell proliferation on DPS. To perform this analysis, the cells were fixed using 4% v/v paraformaldehyde and stained with Phalloidin 488 (Life Technologies, USA) for F-actin, as well as Hoechst 33342. Subsequently, the samples were subjected to imaging using a Nikon A1RHD25 confocal microscope at ×10 magnification. The alignment and orientation of the cells were determined using the OrientationJ plugin within ImageJ, which visualises cell alignment by assigning similar colours to cells with similar orientations. The orientation distribution was generated in the form of a histogram, representing the distribution of cell alignment angles. To quantify the average alignment, the Matlab CircStat toolbox was utilised, resulting in the calculation of a Kappa value within the range of 0-1.

Reagents and antibodies for immunofluorescence and staining of mammalian cell lines were purchased as follow: LAMP1 antibody (H4A3-s, Developmental Studies Hybridoma Bank, Iowa State University), Fluorescently conjugated donkey anti-mouse (Jackson Immunoresearch, West Grove, PA), Phalloidin-488, DAPI, and Slow Fade Gold Reagent (Life Technologies, Carlsbad, CA), Tubulin antibody (1:20,000, Neomarkers, Freemont, CA), Cathepsin B antibody (1:500 Santa Cruz Biotechnology, Dallas, TX).