anti cd147  (R&D Systems)

Journal: Cell Reports Medicine

Article Title: De novo GTP synthesis is a metabolic vulnerability for the interception of brain metastases

doi: 10.1016/j.xcrm.2024.101755

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Article Snippet: APC-conjugated anti-human TRA-1-85 (CD147) , Miltenyi Biotec , Cat#130-128-900; RRID:AB_2921968.

Techniques: Virus, Recombinant, Staining, Labeling, Electron Microscopy, Bradford Assay, RNA Sequencing, Gene Expression, Knock-Out, CRISPR, Luciferase, Software, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Engineered CD147-CAR macrophages for enhanced phagocytosis of cancers

doi: 10.1007/s00262-024-03759-6

Figure Lengend Snippet: CRISPR/Cas9-mediated CD147 knockout in the THP-1 cell line. A Schematic diagram of CD147 knockout in the THP-1 cell line using nucleofection (Created by BioRender.com/Mahidol University). B Flow cytometric analysis of CD147 expression in the THP-1 cells before nucleofection, after nucleofection (pre-sorting) and post-sorting

Article Snippet: On day 11 post-nucleofection, the nucleofected cells were stained with human anti-CD147 Alexa Flour 488-conjugated antibody (R&D systems, FAB3195G).

Techniques: CRISPR, Knock-Out, Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Engineered CD147-CAR macrophages for enhanced phagocytosis of cancers

doi: 10.1007/s00262-024-03759-6

Figure Lengend Snippet: Generation of the CD147 CAR lentiviral vectors and lentiviral transduction of CD147 CAR into the CD147 KO THP-1 cells. A Schematic representation of the CGW CD147 CAR plasmid vector and lentiviral vector production. LTR, Long terminal repeat; MND, a modified enhancer/promoter of the murine myeloproliferative sarcoma virus promoter; CAR, chimeric antigen receptor; sp, signal peptide; scFv, single chain fragment variable; TM, transmembrane domain; Co-SD, co-stimulatory signaling domain; mEGFP, monomeric enhanced green fluorescent protein (Created by BioRender.com/Mahidol University). B Representative fluorescent microscopic images of the WT THP-1 and CD147 CAR-transduced THP-1 cells on day 2 post-transduction, scale bar = 200 µm. C Flow cytometric analysis of the CD147 CAR-transduced THP-1 cells on day 5 pre- and post-sorting. D Representivative confocal microscopy images demonstrating the binding of the CD147 scFv molecules to the recombinant CD147Rg in the WT THP-1 cells and the CD147 CAR THP-1 cells. (63 × oil immersion objective of the Zeiss LSM 800 confocal microscope, scale bar = 10 µm)

Article Snippet: On day 11 post-nucleofection, the nucleofected cells were stained with human anti-CD147 Alexa Flour 488-conjugated antibody (R&D systems, FAB3195G).

Techniques: Transduction, Plasmid Preparation, Modification, Virus, Confocal Microscopy, Binding Assay, Recombinant, Microscopy

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Engineered CD147-CAR macrophages for enhanced phagocytosis of cancers

doi: 10.1007/s00262-024-03759-6

Figure Lengend Snippet: Phagocytosis of zymosan bioparticles. A Schematic diagram illustrating the phagocytosis process of zymosan bioparticles by the wild-type (WT), CD147 KO, and CD147 CAR-macrophages (Created by BioRender.com/Mahidol University). B Representative fluorescent microscopic images depicting the WT, CD147 KO, CD147 CAR macrophages co-cultured with the zymosan bioparticles, scale bar = 100 µm. C Representative histograms and D Bar graph showing phagocytic activity against zymosan bioparticles as determined by flow cytometry. Statistical significance was calculated using multiple unpaired t test analyses, ns indicates P > 0.05. Data are results from three independent experiments

Article Snippet: On day 11 post-nucleofection, the nucleofected cells were stained with human anti-CD147 Alexa Flour 488-conjugated antibody (R&D systems, FAB3195G).

Techniques: Cell Culture, Activity Assay, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Engineered CD147-CAR macrophages for enhanced phagocytosis of cancers

doi: 10.1007/s00262-024-03759-6

Figure Lengend Snippet: Flow cytometric analysis of THP-1 after polarization toward M1 and M2 subtypes. A Representative flow cytometric analysis of the WT, CD147 KO, and CD147 CAR THP-1 cells after activation with PMA (M0, grey), and polarized into M1 (orange) and M2 (green) macrophages. B Mean fluorescence intensity (MFI) of CD80, HLA-DR, CD163, and CD206 of THP-1 cells after polarization. Surface markers include M1 markers (CD80 and HLA-DR), and M2 markers (CD163 and CD206). Statistical significance was calculated using multiple unpaired t test analyses, ns indicates P > 0.05. Data represent the results from three independent experiments

Article Snippet: On day 11 post-nucleofection, the nucleofected cells were stained with human anti-CD147 Alexa Flour 488-conjugated antibody (R&D systems, FAB3195G).

Techniques: Activation Assay, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Engineered CD147-CAR macrophages for enhanced phagocytosis of cancers

doi: 10.1007/s00262-024-03759-6

Figure Lengend Snippet: Phagocytosis of cancer cells. A Flow cytometric analysis of CD147 expression in cancer cell lines, including MCF-7, MDA-MB-231, and K562, compared to the isotype control. B Schematic diagram demonstrating the co-culture experiment of the WT, CD147 KO, and CD147 CAR-M against cancer or normal cells (Created by BioRender.com/Mahidol University). C Live-cell time-lapse phagocytosis assay of the WT and CD147 CAR-M against cancer cell lines captured at 1-h intervals for 24 h. The average total red areas per image (µm 2 ) are plotted. D Representative fluorescent images illustrating the phagocytosis of cancer cells by the WT and CD147 CAR-M in each cancer cell line, scale bar = 100 µm. E The comparison of the red area per image (µm 2 ) at 24 h of co-culture. F TNF-⍺ and IL-6 secretion by the WT and CD147 CAR-M after co-culturing with different cancer cell lines. Statistical significance was calculated using multiple unpaired t test analyses, ns indicates P > 0.05, **indicates P < 0.01, ***indicates P < 0.001. Data represent the results from three independent experiments

Article Snippet: On day 11 post-nucleofection, the nucleofected cells were stained with human anti-CD147 Alexa Flour 488-conjugated antibody (R&D systems, FAB3195G).

Techniques: Expressing, Control, Co-Culture Assay, Phagocytosis Assay, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Engineered CD147-CAR macrophages for enhanced phagocytosis of cancers

doi: 10.1007/s00262-024-03759-6

Figure Lengend Snippet: Phagocytosis of normal cells. A Flow cytometric analysis of CD147 expression in normal cells, including PBMCs (CD4, CD8, CD56, and CD14 subsets), and MCF10A cell line compared to the isotype control. B Live-cell time-lapse imaging of phagocytosis by the WT macrophages and CD147 CAR-M targeting normal cells, recorded at 1-h intervals over 24 h. The graph displays the average total red areas per image (µm 2 ) for each condition. C The red area per image (µm 2 ) at 24 h of co-culture. D TNF-⍺ and IL-6 secretion by the WT macrophages and CD147 CAR-M after co-culturing with PBMCs and MCF10A cells. Statistical significance was calculated using multiple unpaired t test analysis, ns indicates P > 0.05. Data are results from three independent experiments

Article Snippet: On day 11 post-nucleofection, the nucleofected cells were stained with human anti-CD147 Alexa Flour 488-conjugated antibody (R&D systems, FAB3195G).

Techniques: Expressing, Control, Imaging, Co-Culture Assay

Journal: Nature

Article Title: In vitro reconstitution of epigenetic reprogramming in the human germ line

doi: 10.1038/s41586-024-07526-6

Figure Lengend Snippet: a , Scheme for hPGCLC expansion culture (left) and flow cytometric plot for BTAG expression of the hPGCLC culture and for forward and side scatter (FSC and SSC) of the non-BTAG cells (middle). The P1 cells in the middle panel are TRA-1-85 + (a human-specific antigen) , i.e., de-differentiated hPGCLC-derived cells, whereas a majority of the P2 cells are TRA-1-85 − , i.e., m220 feeders (right). Accordingly, the enrichment score is defined as log 2 (the number of BT + AG + cells/the number of cells in the P1 gate) (right). hPGCLCs were cultured as in . See Fig. for the summary of acronyms used in this study. b − d , hPGCLC expansion and the enrichment score of the hPGCLC culture with IWR1 , A83-01, and LDN193189 at culture day (c) 10 and 20 ( b ), with different doses of IWR1 at c10, 20, and 30 ( c ), and with different basal media ( d ). The passages were performed using flow cytometry. The color coding is as indicated. hPGCLCs were cultured as in with or without indicated chemicals. 1 biological replicate for ( b ) and ( d ), and 3 biological replicates for ( c ). e , hPGCLC expansion and the enrichment score of the hPGCLC culture with IWR1 (1.5 μm) in DMEM or advanced RPMI at c12 and 22 (top), and FACS plots for BTAG expression and FSC/SSC of the non-BTAG cells of the hPGCLC culture with IWR1 (1.5 μm) in DMEM or advanced RPMI at c22 (bottom). The passages were performed with dilution. The color coding is as indicated. Note that there were nearly no de-differentiated cells in the P1 gate in the culture with advanced RPMI. The data show (top)/represent (bottom) 2 biological replicates. f , Principal component analysis (PCA) of transcriptomes of key cell types during hPGCLC induction and hPGCLC differentiation in xrOvaries (top) and the identification of genes making significant contributions [radius of standard deviations (SDs) ≥ 3] to scaled PC1 and PC2 loadings (bottom). Genes expressed in at least one sample [log 2 (RPM + 1) ≥ 4] were used for PCA. g , (left) Unsupervised hierarchical clustering (UHC) of the genes selected in ( f ) based on their expression dynamics, and (right) promoter methylation dynamics of the genes in the five clusters in (left) during hPGCLC induction and hPGCLC differentiation in xrOvaries . Among the cluster 2 genes, those showing promoter 5mC-level reduction from human iPS cells (hiPSCs) to oogonia-like cells by ≥ 50% are defined as e pigenetic r eprogramming-activated genes (ER genes). h , Expression (top) and promoter methylation (bottom) dynamics of epigenetic reprogramming-activated genes (ER genes) during hPGCLC differentiation in xrOvaries. Top eight ER genes in the expression level at ag35, and DAZL and DDX4 are annotated. i , Scheme for the screening of cytokines/chemicals that induce ER gene up-regulation. j , Expression of PRDM1 , GTSF1 , PRAME , and MEG3 measured by qRT-PCR at culture day (c) 22 with the indicated cytokines/chemicals. For each gene, ∆Ct from the average Ct values of two housekeeping genes, RPLP0 and PPIA (set as 0), were calculated and plotted for 2 biological replicates. Mean values are shown as a red bar. *, **: Not detected or ∆Ct <−10 in one or two replicates, respectively. ag77: expression values in hPGCLC-derived cell at ag77 in xrOvaries .

Article Snippet: Collected cells were resuspended in 1% BSA–PBS(−) containing anti-TRA-1-85 antibody (BD Bioscience, 563302, 1:20) and placed on ice for 30 min in the dark.

Techniques: Expressing, Derivative Assay, Cell Culture, Flow Cytometry, Methylation, Quantitative RT-PCR