biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsHasan Alrefaiproteomic profiling by arrayHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15227A comparative analysis of 20 analytes from matched primary macrophages polarized to various states was performed to characterize a novel polarization method and compare it to classically polarized M0, M1, and M2 macrophages. Macrophages polarized by the xenoline JX14P and its matched in vivo radiation-selected variant, JX14P-RT, exhibited cytokine expression profiles distinct from M0, M1, and M2 macrophages. Furthermore, macrophages polarized by JX14P exhibit distinct cytokine profiles compared to those polarized by the matched RT-selected xenoline JX14P-RT.biostudies-arrayexpressScaning - Differential cytokine expression analysis A RayBiotech GS1 Cytokine Array Kit (#GSH-CYT-1-4, RayBiotech, USA) was used to quantify the relative abundance of 20 distinct cytokines in the conditioned media of macrophages from 4 biological donors polarized toward 5 different states in technical triplicate. The assay was performed according to the manufacturer’s protocol. Slides were imaged using a GenePix 4100A Microarray Scanner (Molecular Devices, USA) with a 532 nm excitation laser and a PMT gain of 500. GenePix Pro software (v7.4.0) was used to extract and quantify the background-subtracted fluorescence intensities (F532 Mean – B532) for each cytokine.Growth Protocol - PBMC isolation and macrophage differentiation Up to 15 mL of Fresh whole blood from healthy donors was collected in EDTA tubes and diluted with 15 mL phosphate-buffered saline (PBS) in a 50 mL conical tube. 10 mL of Lymphoprep (#18060, StemCell Technologies, Canada) was carefully layered underneath using a 10 mL serological pipette. The tubes were centrifuged at 650 x g for 30 minutes with no brake. The buffy coat was collected and transferred to a new 50 mL tube containing RPMI media (#A1049101, Gibco, USA), then centrifuged for 5 minutes at 650 x g to pellet the cells. The pellet was washed twice with PBS and plated in a 15 cm2 dish with RPMI 1640 supplemented with 10% matched donor serum and 50 ng/mL human M-CSF (#11792-HNAH1, SinoBiological, China). The media was replenished with 50 ng/mL of M-CSF on days 3 and 5. On day 7, the cells were washed three times with PBS and lifted for downstream experiments. After replating, the cells were cultured in a 1:1 mixture of PDX and X-VIVO media supplemented with 25 ng/mL M-CSF.Labeling - n/a. This is a proteomic array. The requested protocol is not relevant for this section.Nucleic Acid Extraction - n/a. This is a proteomic array. The requested protocol is not relevant for this section.Hybridization - n/a. This is a proteomic array. The requested protocol is not relevant for this section.Sample Collection - Conditioned media generation 5x105 macrophages were polarized toward various states. Afterwards, they were washed twice with PBS. 2.2 mL of fresh 1:1 PDX:X-VIVO media supplemented with 25 ng/mL M-CSF was added to each well and the cells were incubated for 24 hours. Afterwards, the media was collected, centrifuged at 1000 x g for 5 minutes, and filtered through a 0.2 µm filter (#09-719C, Fisher Scientific, USA). The resulting conditioned media was divided equally into two portions. The portion used for the cytokine array was spiked with Halt Protease inhibitor (#1861279, ThermoFisher, USA) Phosphatase inhibitor (#P5726-5ML, Sigma-Aldrich, USA), and Halt Phosphatase inhibitor (#1861277, ThermoFisher, USA) at the manufacturers’ suggested concentrations. The other portion was left untreated.Sample Treatment - Macrophage polarization M1 polarization. Resting macrophages were stimulated with 20 ng/mL IFNγ (#300-02-500UG, Gibco, USA) and 1 µg/mL of lipopolysaccharide (#00-4976-03, ThermoFisher, USA) for 24 hours to induce a pro-inflammatory phenotype. M2 polarization. Resting macrophages were stimulated with 20 ng/mL of IL-4 (204-IL, R&D Systems, USA) and IL-13 (#213-ILB, R&D Systems, USA) for 72 hours to induce an anti-inflammatory phenotype. TAM polarization. Resting macrophages were indirectly co-cultured with GBM PDX xenolines using transwell inserts with 3 µm pores (#353096, Corning, USA) for 72 hours, as previously described [45]. Macrophages were seeded in the lower well and GBM was seeded in the upper insert.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsHasan AlrefaiData Transformation - The resulting data was tabulated and exported to R (v4.3.2) for analysis. Raw cytokine array data were processed by normalizing signal intensities to the POS2-control on each slide to account for slide-specific effects. The LIMMA (v3.58.1) package was used to identify differentially expressed (DE) cytokines. A design matrix was constructed using the model.matrix function, followed by fitting a linear model with lmFit(). Pairwise contrasts were defined with makeContrasts() and applied to the model using constrasts.fit() and eBayes() to moderate variance. DE cytokines for all contrasts were extracted using topTable(), with p values adjusted by FDR method (q). Significantly differentially expressed cytokines, q < 0.05, are displayed on volcano plots using the EnhancedVolcano package (v1.20.0). Heatmaps were generated using ComplexHeatmap (v2.18.0) on log2(n+1) transformed normalized intensities. PCA was performed using PCAtools (v2.14.0).falseGS1 Cytokine Array Analysis of Primary Human Macrophages Polarized to M0, M1, M2, 14P-TAM, and 14P-RT TAM statesA comparative analysis of 20 analytes from matched primary macrophages polarized to various states was performed to characterize a novel polarization method and compare it to classically polarized M0, M1, and M2 macrophages. Macrophages polarized by the xenoline JX14P and its matched in vivo radiation-selected variant, JX14P-RT, exhibited cytokine expression profiles distinct from M0, M1, and M2 macrophages. Furthermore, macrophages polarized by JX14P exhibit distinct cytokine profiles compared to those polarized by the matched RT-selected xenoline JX14P-RT.2025-12-31T00:00:00Z2025-12-31T02:02:47.8Z2025-06-13T14:50:19.767ZE-MTAB-15227EFO_0003814EFO_0002944EFO_0003813EFO_0003789EFO_0002765EFO_0005518EFO_0003816EFO_0003969EFO_0003815