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?0.05; **p?0.01; ***p?0.001; ****p?0.0001. Brightfield image analysis is a robust normalization technique for PBMCs To validate if brightfield image analysis is a robust method for XF assay data normalization, we studied the effect of PIXI analysis normalization on the accuracy and precision of XF assay parameter determination. The accuracy of brightfield image analysis was tested by assessing if individual observations, i.e. technical replicate values, were closer to the overall group mean after transforming the number of plated cells to the number of PIXI analyzed cells, using standard scores. Standard scores that come closer to zero indicate that individual observations are better PF-04457845 predictors of the overall mean and therefore correspond to a higher level of accuracy. Normalization to the number of PIXI analyzed cells decreased the average standard score of initial OCR (from 0.694 to 0.663), basal OCR (from 0.684 to 0.654) and basal GR (from 0.675 to 0.668) (Fig.?5a and Table ?Table1),1), indicating that PIXI analysis improved the accuracy of mean XF assay parameter estimation between technical replicates. To test the precision of brightfield image analysis, the effect of PIXI analysis on the distribution of standard scores was calculated via calculation of standard score interquartile ranges (IQR). If IQRs are smaller, individual standard scores deviate less from the mean standard scores, and therefore correspond to a higher level of precision. Normalization to the number of PIXI analyzed cells lowered the average IQR of initial OCR (from 1.520 to 1 1.332), basal OCR (from 1.435 to 1 1.297) and basal GR (from 1.499 to 1 1.382) (Fig.?5b and Table ?Table1)1) which indicated that PIXI analysis resulted in higher levels of precision when estimating mean XF assay parameters between technical replicates. Overall, these results indicated that PIXI analysis resulted in accurate and precise estimation of XF assay parameters, and can therefore be considered as a robust normalization technique. Open in a separate window Figure 5 The effects of PIXI analysis normalization on the standard scores and standard score interquartile ranges (IQR). Mean standard scores and standard score interquartile ranges of all technical replicates within each cell density (50C300??103 cells/well, n?=?14C16) were averaged and used to create an overall average standard score and standard score IQR for each XF assay parameter (N?=?18) as a measure of accuracy (standard score) and precision (IQR). (a,b) Effects of brightfield image PIXI analysis normalization on the average standard score (a) or standard score IQR (b) in PF-04457845 initial OCR, basal OCR and basal GR (not significant). Table 1 Summary of the changes in average standard.