Principal components analysis was performed using the command prcomp from the R stats package (87), and plots were produced with the ggbiplot package (88). Data were z-normalized, and missing values were inputted with the mean of all valid values for that metabolite. Colocalization and fluorescence quantification analysis were performed on ImageJ using Manders’ analysis and Costes’ automatic threshold (8991). Five images were acquired from each mouse with three mice per condition. PD-L1 image stacks were collapsed to 2D using the maximum intensity projection, and the black level was subtracted from all pixels. All pixels less than gray level 1000 were removed to mask out nonspecific signal. The mean of the remaining pixels was calculated to represent each image. CD31 images were quantified by manually counting macroscopic vascular structures per field of view. Graphics and statistical analyses for all figures were performed using GraphPad Prism software. All results represent means ± SEM, unless stated otherwise. The significance of differences between the means or the population distributions was determined using two-way analysis of variance (ANOVA) test (for metabolomics, live imaging analysis, and PD-L1 immunofluorescence quantification), two-tailed unpaired Student’s t test (for RT-qPCR, Western blot, ELISA, and colorimetric assays), or nonparametric Mann-Whitney test (for CD31 IF quantification). For all tests, differences were considered statistically significant if P values were <0.05, 0.01, and 0.001 (indicated with *, **, and ***, respectively, in the figures).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.