Varioskan plate reader

50 μg of the membrane protein fraction overexpressing GFP or RPTPσ N/C was incubated with different amount of Chondroitin sulfate or Heparin at 37°C for 30 minutes followed by the addition of 1 mM p-nitrophenyl phosphate for 30 min. The assay was conducted at 25°C in 96 well plates and the absorbance of p-nitrophenol was monitored at 405 nm every minute using a Varioskan plate reader (Thermo Electron).

The cytotoxicity of TBE-50 and TBE-70 in adipocytes was evaluated by the WST-8 [2-(2-methoxy-4-nitropheyl)-3-(4-nitrophenyl)-5-(2,4-dinitrophenyl)-2H-tetrazolium, monosodium salt method, using a commercial CCK-8 kit as described previously [63 (link)]. Differentiated 3T3-L1 adipocytes were incubated for 1, 2, 5, or 7 days with TBE-50 or TBE-70 (at a dose of 0, 0.1, 1, 10, 50, 100, or 500 µg/mL). The absorbance was read using a Varioskan plate reader (Thermo Electron, Waltham, MA, USA) at 450 nm, and the results are expressed as the percentage of the control. All assays were carried out in triplicate.

Cell viability was determined as described previously [16 (link)] using a CCK-8 kit according to the manufacturer’s instructions. Differentiated 3T3-L1 adipocytes were treated with 0 (control), 0.1, 1, 10, 100, or 1000 ng/mL of SN or AN for 4 and 24 h at 37°C. For the negative and positive control samples, 0.1% dextran and 0.1% sodium dodecyl sulfate (SDS) was added, respectively. Absorbance was measured using a Varioskan plate reader at 450 nm (Thermo Electron, Waltham, MA, USA) and results are presented as the percentage of untreated control cells. All measurements were performed in at least three independent experiments, each of which was performed in triplicate (n = 3).

The cytotoxicity of ISOR in adipocytes was evaluated using the WST-8 [2-(2-methoxy-4-nitropheyl)-3-(4-nitrophenyl)-5-(2,4-dinitrophenyl)-2H-tetrazolium, monosodium salt method, with a commercial CCK-8 kit as described previously [37 (link)]. Differentiated 3T3-L1 adipocytes were incubated for 1, 2, 5, or 7 days with ISOR (at a dose of 0, 0.1, 0.5, 1, 10, 20, or 50 µM). The absorbance was read using a Varioskan plate reader (Thermo Electron, Waltham, MA, USA) at 450 nm, and the results are expressed as a percentage of the control. All assays were carried out in triplicate.

ANS (Sigma, St. Louis, MO, USA) is a lipophilic reagent that incorporates naturally into membranes and fluoresces at 512 nm when excited at 350 nm. The ANS assay was performed in parallel on liposomes (0.86 mM phosphatidyl choline (PC), 0.24 mM phosphatidyl ethanolamine (PE) and 0.24 mM cardiolipin (CL)) and OMVs, liposomes serving as references of the ANS assay, as described earlier [23 (link)]. An increasing concentration in liposomes (1 to 5 µg/µL in Tris-KCl buffer (50 mM Tris-HCl, 200 mM KCl, pH 7.5)) as well as 1 or 2 µL of each OMV solution were incubated with 200 µL of 0.001% ANS (in Tris-KCl buffer) in a black flat-bottom 96-well plate (Corning, Corning, NY, USA). The fluorescence was measured at 512 nm after excitation at 350 nm for 100 ms using a Varioskan plate reader (Thermofisher). Concentration measurements were performed in triplicates, and the mean concentrations in lipids were calculated and used to quantify the OMVs in the experiments involving OMVs. The lipid concentration in REL606 OMVs was found to be 2 mg/mL, while that of miRFP713-OMVs was 0.7 mg/mL.