Ts2r microscope

LLC-PK1 cells were grown in the 4-well Nunc™ Lab-Tek II CC2™ chamber slide system (Thermo Fisher Scientific, Somerset, NJ, USA; Cat #154917) and treated for 48 h. After treatment, cell morphology was examined under the Nikon (Tokyo, Japan) Ts2R microscope and micrographs were captured. The cell monolayers were then fixed with 4% paraformaldehyde for 15 min at room temperature followed by permeabilization with 0.1% Triton X-100 for 10 min. Monolayers were blocked with 2% w/v bovine serum albumin (BSA) in PBS containing 0.3 M glycine for an hour and then incubated overnight at 4 °C with the primary mouse monoclonal antibodies against α-SMA (1:100), vimentin (1:500) or β-catenin (1:100). Monolayers were then washed with ice-cold PBS, incubated with FITC-labeled anti-mouse IgG antibody (1:1000) for 1 h, then washed thrice with PBS. After the final wash, the slides were mounted in Prolong™ diamond anti-fade reagent with DAPI (Thermo Fisher Scientific, Somerset, NJ, USA; Cat #P36962) and observed under a Nikon (Tokyo, Japan) Ts2R microscope.

Autoreactivity was determined by ANA Hep-2 Staining Analysis (ZEUS Scientific Cat. No: FA2400) and anticardiolipin ELISA (Inova Diagnostics Cat. No.: 708625). For the Hep-2 assay, all antibodies were tested at 25 and 50 μg/ml as per manufacturer’s protocol and imaged on a Nikon Ts2R microscope for 500 ms. Scores from 0 to 3 were defined with four control antibodies VRC01-LS, 4E10, VRC07-523LS, and VRC07-G54W. Test antibodies were scored by visual estimation of staining intensity in comparison to the control antibodies. Scores equal to or greater than 1 at 25 μg/ml were classified as autoreactive, and between 0 and 1 as mildly autoreactive. In the cardiolipin ELISA, antibodies were tested at a starting concentration of 100 μg/ml, followed by threefold dilutions. IgG phospholipid (GPL) units were calculated from the standard curve. GPL score < 20 was considered as not reactive, 20–80 as low positive and > 80 as high positive.