Confocal Analysis of hGMSCs Signaling

To perform the Confocal Laser Scanning Microscope analysis, 6.4 × 10³/well of hGMSCs were placed in 8-well culture glass slides and treated with different stimuli for 72 h (as mentioned above). Then, the cells were fixed with 4% of paraformaldehyde (PFA) (BioOptica, Milan, Italy) in PBS (0.1 M) (Lonza, Basel, Switzerland) for 1 h at room temperature, washed 3 times with PBS, permeabilized with 0.1% Triton X-100 (BioOptica) in PBS and blocked for 1 h at room temperature with 5% of skimmed milk in PBS. The primary antibodies anti-TLR4 (sc-293072, Santa Cruz Biotechnology, Dallas, TX, USA), anti-MyD88 (sc-74532, Santa Cruz Biotechnology), anti-NFκB p65 (sc-8008, Santa Cruz Biotechnology) and anti-NALP3 (sc-134306, Santa Cruz, Biotechnology) were diluted 1:200 and incubated overnight at 4 °C. Then, the secondary antibody Alexa Fluor 568 red fluorescence-conjugated goat anti-mouse (1:200) (A11031, Invitrogen, Eugene, OR, USA) was added to the samples for 1 h at room temperature, followed by the final incubation with 1:200 of Alexa Fluor 488 phalloidin green fluorescent conjugate (A12379, Invitrogen) and TOPRO (T3605, Invitrogen) mixed together. The images were obtained with a Zeiss LSM800 confocal system (Carl Zeiss, Jena, Germany) [49 (link)].