Alexa fluor 680 phalloidin

MIC-1 (purity >98%) was isolated from Moringa oleifera seed extract as previously reported [1 (link)]. All other materials were purchased from commercial suppliers. C2C12 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). C57BL/6 male mice were acquired from Charles River Laboratories (Malvern, PA, USA). Cell culture materials: Dulbecco’s modified Eagle’s medium (DMEM), penicillin G, streptomycin, and fetal bovine serum (FBS) were obtained from Gibco Inc. (Grand Island, NY). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Cayman Chemical (Ann Arbor, MI, USA) and Griess reagent system was acquired from Promega Corporation (Madison, WI, USA). Lipopolysaccharide (LPS, Escherichia coli 0111: B4), dimethyl sulfoxide (DMSO), and other chemicals were purchased from Sigma (St. Louis, MO, USA). Gene probes were obtained from Integrated DNA Technologies (Coralville, IA, USA), and the probe sequences are the same as our previous study [8 (link)]. RNeasy Mini Kit was obtained from Qiagen (Germantown, MD, USA). Anti-NF-κB p65 antibody (ab16502) was obtained from Abcam (Cambridge, MA, USA), and 4′,6-diamidino-2-phenylindole (DAPI) and Alexa Fluor™ 680 phalloidin were purchased from Invitrogen (Carlsbad, CA, USA).

To visualise OMV entry into host cells, Caco-2 cells were seeded on 18mm round coverslips (Marienfeld, Germany) in 12-well plates at a density of 3 × 10⁵ cells per well in 1ml of media for 24 h. Caco-2 cells were stimulated with either DiI, BODIPY-FL or Syto RNASelect-labelled B. fragilis OMVs for 4 h at an MOI of 4 × 10⁵ OMVs per cell, or each respective stain in DPBS as a control. Following incubation, cells were washed three times with DPBS, and extracellular fluorescence was quenched with 0.025% (v/v) Trypan blue as previously described (34 (link)). Cells were fixed using 4% paraformaldehyde (Sigma-Aldrich, USA) and blocked using 1% BSA (w/v) in DPBS. Cell nuclei and cellular actin were stained with 4’,6‐diamidino‐2‐phenylindole dilactate (DAPI; Merck, Germany) and Alexa Fluor 680 phalloidin (Invitrogen, USA), respectively. Samples were then mounted using VectaShield mounting medium (Vector Laboratories, USA) and imaged using a Zeiss 780 PicoQuant confocal microscope (Zeiss, Germany) using a 63x/1.4NA oil objective at 1024 × 1024 × 32 bit per channel. Image analysis was performed using Imaris x64 v9.5.0 (Bitplane, Switzerland). Three biological replicates of Caco-2 cells stimulated with OMVs, labelled with each individual stain, were examined. Three fields of view were imaged for each treatment containing a minimum of 10 cells per field of view.