qPCR analysis.

<p>Heat map showing qPCR analysis results of selected genes in various passages of RAW264.7 cells. Differences in gene expression are represented by ΔΔCt values of three biological and three technical replicates. The control for this experiment constituted cells from the passage no. 5. Their gene expression value equals 1. Analysis was performed using R (<a href="https://cran.r-project.org/" target="_blank">https://cran.r-project.org</a>) package gplots [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198943#pone.0198943.ref025" target="_blank">25</a>].</p

Phagocytosis assay.

<p>Results of phagocytosis assay performed on RAW 264.7 cells at various passages. Graphs A, C, E show percentage of phagocyting cells, positive for FITC fluorochrome conjugated with latex beads. Graphs B, D, F show the intensity of phagocytosis expressed as mean fluorescence intensity of latex beads. A, B–phagocytosis of RAW 264.7 cells. C, D–phagocytosis of LPS treated RAW 264.7 cells. E, F–comparison of phagocytosis ability of RAW 264.7 cells in various condition, ice control indicating technical control for phagocytosis assay. *- p-value < 0.05, **—p-value < 0.01.</p

Flow cytometry analysis.

<p>Flow cytometry analysis of CD11b, CD11c, CD86, CD200R, CXCR4 and Ly6C surface markers expression on RAW 264.7 cells. Values of expression are expressed as geometric mean of MFI (Mean Fluorescence Intensity). *- p-value < 0.05, **—p-value < 0.01.</p

Nitrogen level in cell culture medium.

<p>Level of nitrogen in cell culture medium of RAW 264.7 cells at various passages. A—comparison of NO level in control cells and LPS stimulated cells. B– 24 hrs production of nitric oxide by LPS stimulated RAW 264.7 cells. Data presented as average concentration of produced nitrites, calculated on the standard curve basis. *—p-value < 0.05, **—p-value < 0.01, ***—p-value < 0.001, ****—p-value < 0.0001.</p