Image quality was checked based on visual inspection of the pictures. Brightfield image analysis in R Original brightfield images were processed and quantified using an in-house generated R PF-04457845 script that used the EBImage package available in Bioconductor76 and included four steps. assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the Ngfr standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood PF-04457845 cells instead of immortalized cell lines for immunometabolic experiments. not significant; *p?PF-04457845 initial OCR, basal OCR and basal GR (not significant). Table 1 Summary of the changes in average standard.