Alexa fluor 488 phalloidin green fluorescent conjugate

Sample fixation was performed with a solution of 4% of paraformaldehyde in 0.1 M of PBS (Lonza) [35 (link), 36 (link)]. The following steps were performed: cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min; samples blocking with 5% skimmed milk in PBS for 30 min [37 (link)]; primary antibodies (anti-NFκB, 1 : 200, Santa Cruz Biotechnology; anti-MyD800, Thermo Fisher Scientific; anti-DNMT1, 1 : 200, EpiGentek; and anti-p300, 1 : 200, OriGene) incubation for 2 h at room temperature; and finally, secondary antibody (Alexa Fluor 568 red fluorescence-conjugated goat anti-rabbit antibody, 1 : 200, Molecular Probes, Invitrogen, Eugene, OR, USA) incubation for 1 h at 37°C. Cells were stained for 1 h with Alexa Fluor 488 phalloidin green fluorescent conjugate (1 : 400, Molecular Probes) and for 1 h with TOPRO (1 : 200, Molecular Probes) to mark the cytoskeleton actin and nuclei, respectively [38 (link), 39 (link)]. The Zeiss LSM800 confocal system (Zeiss, Jena, Germany) has been used to acquire microphotographs.

The hDPSCs were fixed using 4% paraformaldehyde solution in sodium phosphate buffer (Lonza, Basel, Switzerland) [53 (link),54 (link)]. The cells were permeabilized utilizing 0.5% Triton X-100 in PBS for 10 min followed by blocking with 5% skimmed milk in PBS for 30 min [55 (link)]; primary antibodies (anti-NFkB antibody, 1:250, Santa Cruz Biotechnology; anti-ERK antibody,1:200, Santa Cruz Biotechnology; anti-pERK antibody,1:200, Santa Cruz Biotechnology) were incubated for 2 h at room temperature. At the end of incubation cells were processed using secondary antibody (Alexa Fluor 568 red fluorescence conjugated goat anti-rabbit antibody, 1:200, Molecular Probes, Invitrogen, Eugene, OR, USA) incubation for 1 h at 37 °C. To depict the cytoskeleton actin, samples were treated with the Alexa Fluor 488 phalloidin green fluorescent conjugate (1:400, Molecular Probes, Eugene, OR, USA) for 1 h [56 (link)]. After washings, cells were incubated with TOPRO (1:200, Molecular Probes) for 1 h at 37 °C [57 (link)] to stain cell nuclei. Samples were detected using a Zeiss LSM800 confocal system (Zeiss, Jena, Germany).

Samples were fixed with 4% paraformaldehyde in 0.1 M of PBS (Lonza, Basel, Switzerland). Then, cells were permeabilized with 0.5% Triton X-100 in PBS (Lonza, Basel, Switzerland) for 10 min and blocked with 5% skimmed milk in PBS for 30 min [27 (link)]. The following primary antibodies were used in the study: anti-TLR4 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-MyD88 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NFκB (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NLRP3(1:500; Novus, Centennial, CO, USA), anti-Caspase-1 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-IL-1β (5 µg/mL; ThermoFisher, Waltham, MA, USA). Cells were incubated with primary antibody for 2 h at room temperature. Then, samples were incubated with Alexa Fluor 568 red fluorescence conjugated goat anti-rabbit secondary antibody (1:200; Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h at 37 °C. To stain the cytoskeleton actin, cells were treated with Alexa Fluor 488 phalloidin green fluorescent conjugate (1:400, Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h, and to stain the nuclei, cells were stained with TOPRO (1:200; Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h. The Zeiss LSM800 confocal system (Zeiss, Jena, Germany) was used to acquire microphotographs.