S8032

Cells were centrifuged and stained for 30 mins at RT with Live/Dead UV Blue stain (Life Technologies, # L23105; diluted 1:1000 in PBS) for assessing viability. After washing, cells were exposed to Fc receptor block (50 μg of unconjugated human IgG; Sigma Aldrich #S-8032) and stained for surface proteins (30 mins at 4°C) by adding respective antibodies in FACs buffer. The FACS buffer is Ca⁺²/Mg⁺²-free PBS with 1% heat-inactivated fetal bovine serum (Sigma Aldrich #F2442) and 0.02% sodium azide (Sigma Aldrich #S-8032). For analyzing intracellular proteins, the cells were then subjected to fixation and permeabilization according to the manufacturer’s guidelines (eBioscience, San Diego CA, USA, #00-5123-43, #00-5223-56 and #00-8333-56; BD Biosciences #51-2090KZ, #51-2091KZ) followed by staining with appropriate antibodies. Flow cytometry data was acquired on Fortessa (BD Bioscience) and analyzed with FACSDiva software (BD Biosciences) or Flowjo.

Bovine serum albumin (BSA; Roche Diagnostics, Basel, Swiss) in a phosphate buffer containing sodium azide (s-8032, Sigma-Aldrich, St. Louis, MO, USA) was added to a 0.2 M solution of glucose and fructose. This solution was added to the OSSCE or aminoguanidine (AG; 396494; Sigma-Aldrich), a positive control. Following 14 days of incubation, the AGE-specific fluorescence was analysed using a spectrofluorometer (Synergy HT; BIO-TEK, Winooski, VT, USA; 370 nm/440 nm). The IC₅₀ (inhibitory concentration which nonenzymatic AGE formation is reduced by half) was calculated from the dose inhibition curve.

Thymidine is interchangeable with 5-ethynyl-2′-deoxyuridine (EdU) and can thus be incorporated into DNA instead of thymidine in the replication process. This can then be used to measure cellular proliferation by detecting how much EdU was inserted into the DNA. Cells were cultured at a cell seeding density of 1 × 10⁵ and incubated for 24 h with fluorescent azide. Following incubation, the iADSCs were labelled with immunofluorescent markers. A 10% solution of paraformaldehyde (P6148, Sigma-Aldrich) was used to fix cells for 10 min. A 2% BSA solution was used to permeabilize cells for 30 min at room temperature (RT); cells were subsequently washed with PBS three times. Cells were then treated with (1 : 200) α actin primary antibody and incubated at RT for 30 min. The treated iADSCs were rinsed three times with ice-cold washing buffer (azide/PBS/BSA: 0.01% w/v sodium azide (Sigma-Aldrich, S8032)), PBS, and 0.1% w/v BSA (Sigma-Aldrich, A2153) three times. Then, cells were incubated with donkey anti-mouse Alexa fluor 594 secondary antibodies (1 : 800) at RT for 30 min. Cells were again treated with washing buffer and counterstained with 300 nm 4′,6-diamidino-2-phenylindole (DAPI). The counterstained cells were mounted with Fluoromount™ onto microscopic glass cover slides and observed with an Axio Observer Z1 (Carl Zeiss) microscope.