Phalloidin ifluor 488 reagent

Cells were grown and treated on coverslips, then fixed with a 4% paraformaldehyde Phosphate Buffer Saline solution and incubated with GPER (Santa Cruz, CA, USA; sc-48525), β-tubulin (Cell Signaling, Danvers, MA, USA; 21285S) primary antibodies (1:100 dilution) followed by incubation with Alexa 555 anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA; A21429) (1:1000 dilution) as previously described [38 (link)] or with phalloidin-i-Fluor 488 reagent (Abcam, Cambridge, UK; Ab176753). Finally, the cells were incubated with 1 µg/mL Hoechst 33342 (Sigma-Aldrich). Images were obtained with an ImageXpress (Molecular Devices, San Jose, CA, USA) microconfocal imager.

After seeding overnight in 35 mm, high µ-dishes (ibidi USA Inc., 81,156), cells were treated as indicated in figure legends, washed twice in PBS and incubated in fixation buffer (3.7% formaldehyde in 100 mM PIPES at pH 6.8, 10 mM EGTA, 1 mM magnesium chloride, and 0.2% Triton X-100) for 10 min at room temperature. Cells were washed as before, treated with Phalloidin-iFluor 488 Reagent (abcam, ab176753) for 1 h in 1–5% BSA in PBS, followed by Hoechst 33342 (Thermo Fisher Scientific, H3570) for 5 min. Mounting medium (Fisher Scientific, P10144) and cover slips were applied onto dried cells. Individually discernible cells were imaged on an Olympus Fluoview 10i. Using ImageJ, the phalloidin channel was thresholded and area and circularity values were measured and integrated into swarmplots using Python’s seaborn library.
For time lapse microscopy, FH-B cells in high µ-dishes were incubated with CellBrite Steady Membrane stain (Biotium, 30108-T) for 30–40 min in the environmental chamber of a Nikon C2 Si microscope at 37 °C and 5% CO₂. Treatments were added to the cells through a syringe to reach final concentrations of 4 µM FQI1 or 0.01% DMSO. Immediately thereafter, a time-lapse series was acquired by imaging every 30 s for 30 min at 20× magnification.

Cultured cells were subjected to Live/Dead staining (consisting of calcein/ethidium; ThermoScientific, Waltham, MA, USA) to check their viability after 72 h in culture on the cryosection dECM hydrogels. Cells were fixed with 4% PFA (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and washed with 1X PBS 3 times.
For F-actin fibroblast were fixed in 4% PFA (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and permeabilized with 0.1% triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min. Subsequently, the samples were incubated with Phalloidin-iFluor 488 Reagent (ab176753, Abcam, Cambridge, UK) at a dilution of 1:750 for 45 min at room temperature. The samples were then washed 3 times with PBS1x and incubated with a 1:1000 solution of DAPI (NucBlue, ThermoScientific, Waltham, MA, USA). The samples were then washed with PBS 3 times and transferred with the seeded surfaces facing down onto a coverslip using Fluoromount-G mounting medium (ThermoScientific, Waltham, MA, USA).
For fluorescence imaging acquisition, a Nikon D-Eclipse Ci confocal microscope was used in conjunction with a ×10 Plan Fluor (Nikon) for Live/Dead staining-(epifluorescence mode) and ×20 Plan Apo immersion oil objective (Nikon) for F-actin staining (z-stack confocal mode).

At 24 h of culturing, samples cultured with osteoblasts and fibroblasts were washed with PBS (VWR^®, Radnor, PA, USA). Cell fixation was performed using 4% formaldehyde solution for 10 min and stained with Phalloidin-iFluor 488 Reagent (ab176753, Abcam^®, The Netherlands) and Propidium iodide (P4179, Sigma-Aldrich^®, St. Louis, MO, USA). Fluorescence images were carried out at 493/517 nm and 535/617 nm wavelength using a Leica TCS SP5 confocal microscope (Leica Microsystems, Deerfield, IL, USA) coupled to LAS-AF LITE v2.0 software (Leica Microsystems, Deerfield, IL, USA).

Cells were washed with PBS, fixed with 4% PFA in PBS for 20 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and blocked for 1 h (3% BSA, 0.1% Triton X-100 in PBS). Incubation with indicated primary antibodies was carried out for 2 h at RT followed by incubation with Alexa Fluor-conjugated secondary antibodies (Abcam) for 30 min at RT. DAPI was used to stain DNA. To visualize F-actin, Phalloidin-iFluor 488 Reagent (Abcam) was used. Slides were mounted with ProLong Gold Antifade Mountant (Invitrogen). Images were acquired using Axio Imager Zeiss 2 (EC Plan—Nefluar objectives) and analyzed with ZEN 2.3 or ImageJ software.

The following chemicals were acquired from Sigma-Aldrich (St. Louis, MO, USA): neodymium (III) chloride hexahydrate (NdCl₃·6H₂O, 99.9%), calcium chloride dihydrate (CaCl₂·2H₂O, 99.5%), cerium (III) chloride heptahydrate (CeCl₃·7H₂O, 99.9%), potassium citrate tribasic monohydrate (HOC(COOK)(CH₂COOK)₂·H₂O, ≥99.0%), gadolinium (III) chloride hexahydrate (GdCl₃·6H₂O, 99.9%), ammonium fluoride (NH₄F, ≥98.0%). Biolegend (San Diego, CA, USA) supplied the anti-CD40-APC. Thermo Fisher Scientific (Waltham, MA, USA) provided fetal calf serum (FCS), 4′,6-Diamidino-2-Phenylindole (DAPI), dulbecco’s Modified Eagle’s Medium (DMEM) and CD86-FITC. Promega (Madison, WI, USA) offered cell titer 96 AQueous MTS Reagent Powder. PeproTech (Cranbury, NJ, USA) supplied lipopolysaccharide (LPS). Bioline (London, UK) delivered agarose. Abcam (Cambridge, UK) supplied the phalloidin- iFluor 488 Reagent. All of water used in the experiments was ultrapure deionized water.