Imagej software

R (ver. 4.2.1) (R Foundation, Austria) and the ImageJ software were used to analyze the data.

Cells (1.5 × 10⁵/well) were seeded into six-well plates in 2 ml EMEM+/+ and incubated for 24 h. The cells were detached with Accutase (Sigma-Aldrich) and washed in PBS−/−, starved for 4 h, and treated with vehicle or 5 μg/ml MPO. A 24-well invasion chamber (#354480 Corning) was used. The chamber was activated by the addition of warm (37 °C) EMEM−/− to the interior of the inserts (0.5 ml) and bottom of the wells (0.5 ml) for 2 h. Upon activation, the medium was removed, and 750 μl EMEM +/+ was added to the bottom well. The treated cell suspension was added to the insert and incubated for 24 h at 37 °C, and 5% CO₂. Afterwards, non-invaded cells were removed from the upper part of the membrane by wiping with cotton swab over the membrane of the chamber. The invaded cells were fixed and stained with Diff-Quick solution. A scalpel was used to remove the membrane from the insert and placed on a microscopy slide. Five photos of each membrane were captured at 100 × magnification using Olympus B×53 with an Olympus UC90 camera. CellSense Standard software was used. ImageJ software (R Foundation) cell counter function was used to count the invaded cells.

Statistical Chi squared test for equality of proportions was applied in order to correlate urinary OspA outcome (detectable or non-detectable) to clinical LB diagnosis and serology. Power calculations were performed in order to estimate the power of the test, given the number of samples in each group, the proportions of urinary OspA outcome, and a significance level of 0.05. Calculations were performed using R software (http://www.r-project.com).
Western blotting band intensity was quantified with imagej software (imagej.nih.gov/ij/index.html">http://imagej.nih.gov/ij/index.html) by selecting the area of interest and calculating area, mean and standard deviation of selection peR software instructions. Blast analysis and protein alignment was performed using pBLAST [32 (link)]. Search parameters were as follows: query sequence: KTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGT, Database Name: non redundant, Program: BLASTP 2.2.31+.