Mfp3d bio instrument

Erythrocyte solutions were deposited onto freshly cleaved mica and an MFP3D-BIO instrument (Asylum Research) used. Samples were nanoindented under RPMI-HEPES with MLCT silicon nitride probes (nominal spring constant 0.1 N/m, resonant frequency 38 kHz, Bruker AFM Probes). To calculate erythrocyte modulus a total of 1000 force curves were registered distributed in at least 20 different points for each condition. Analysis was made by NanomechPro software (Asylum Research).

Fresh DOPC:DOPS (1:1) SUVs were unrolled on freshly cleaved mica (∅ 1.2 cm) in HBS-Ca (10 mM HEPES, 150 mM NaCl, 2 mM CaCl₂, pH 7.5), by covering the mica entirely with 0.2 mg ml⁻¹ SUVs, followed by a 20-min incubation at 30 °C, and washing twice with HBS-Ca.
The samples were imaged immediately in HBS at ambient temperature using an Asylum Research MFP-3D Bio instrument. TR800PSA cantilevers (Olympus; spring constant (k) range 0.59–0.68 N m⁻¹, resonance frequency 77 kHz) were used in alternative current (AC) mode. Square 256 × 256 pixel scans were acquired from areas between 5–20 µm, using a 90° scanning angle and a 0.6–0.8 Hz scan speed. The resulting scan images were processed in Igor Pro 6.37.
After confirming the presence of lipid bilayers, 1.8–10 µM P0ct was added onto the bilayer samples in HBS. After a 15-min incubation period at ambient temperature, the bilayers were washed twice with HBS, and imaged as above. For each protein concentration, 2 samples were prepared and scanned with identical results. At least 3 different parts per sample were scanned to gain an insight into any sample heterogeneity.