Journal: bioRxiv

Article Title: γδ T cells are effectors of immune checkpoint blockade in mismatch repair-deficient colon cancers with antigen presentation defects

doi: 10.1101/2021.10.14.464229

Figure Lengend Snippet: a. Flow cytometry gating strategy on PDTO cells for analysis of surface staining. Selected cells were gated on single, live cells before quantification of staining signal. b. Histogram representation and count for surface staining of MHC-I, PD-L1, and β2m expression on two PDTO lines B2MWT and B2MKO after IFNγ pre-stimulation. Staining with isotype antibodies for each fluorochrome (PE, APC and FITC) were included as negative control. c. Flow cytometry gating strategy on γδ T cell samples for analysis of intracellular staining to test antitumor reactivity upon PDTO stimulation. Lymphocyte population was further gated on single cells, live and CD3+ cells, γδ TCR+ cells and CD8+ as well as CD8–CD4– cells. Reactivity of the sample was based on IFNγ+ cells of the selected population. d. Histogram representation and count for surface staining of NKG2D ligands MICA/B, ULBP1, ULBP2/5/6, ULBP3, and ULBP4 on two PDTO lines B2MWT and B2MKO after IFNγ pre-stimulation. e. Flow cytometry gating strategy on γδ T cell samples for analysis of intracellular staining after stimulation with PDTOs in the presence of NKG2D ligand blocking. Lymphocyte population was further gated on single cells, live and CD3+ cells, followed by γδ TCR+ and CD8+ as well as CD8– cells. Reactivity of final population was based on IFNγ+ or CD107a+ cells.

Article Snippet: Briefly, cells were incubated with human Fc receptor block (BioLegend) and stained with the different cell surface antibodies (1:10 anti-CD112-PE [clone R2.525, BD Biosciences], 1:10 anti-CD155-PE [clone 300907, R&D Systems], 1:50 anti-CD277/BTN3A1-PE [clone BT3.1, Miltenyi], 1:100 anti-HLA-A,B,C-FITC [clone W6/32, eBioscience], 1:20 anti-HLA-E­BV421 [clone 3D12, BioLegend], 1:20 anti-HLA-G-APC [clone 87G, BioLegend], 1:300 anti-MICA/B-PE [clone 6D4, BioLegend], 1:10 anti-ULBP1-PE [clone 170818, R&D Systems], 1:20 anti-ULBP2/5/6-PE [clone 165903, R&D Systems], 1:20 anti-ULBP3-PE [clone 166510, R&D Systems], or 1:20 anti-ULBP4-PE [clone 709116, R&D Systems] for 45 min at 4°C.

Techniques: Flow Cytometry, Staining, Expressing, Negative Control, Blocking Assay

Journal: bioRxiv

Article Title: Highly multiplexed mRNA quantitation with CRISPR-Cas13

doi: 10.1101/2023.08.16.553527

Figure Lengend Snippet: a , Huh7 cells were infected with nine flaviviruses. Total RNA extracted from infected Huh7 cells was used as input for the qCARMEN ISG panel. b , Calculated fold-changes in expression for ISG panel genes across infected cells compared to naive cells. c , The distribution of fold-changes for each gene across viral infections and replicates. Boxplot whiskers extend to 1.5 times the interquartile range; outliers represented with diamond markers. d , Pairwise Spearman correlations of dysregulated ISGs in response to flavivirus infection. e , Gene expression vectors at each timepoint for all three IFNs plotted along two principal components to illustrate the trajectory of expression changes across core ISGs. PCA were transformations performed on flavivirus ISG signatures to overlay the expression profiles on top of IFN stimulation trajectories. Gold star represents 0-hour timepoint. Light to dark: 1, 2, 4, 8, 24, 48, 72-hour timepoints. f , Euclidean distance between flavivirus PCA projections and IFN timepoint vectors to characterize flavivirus ISG changes in the context of IFN responses.

Article Snippet: Pellets were subsequently incubated for one hour at room temperature with flavivirus group antigen antibody (clone 4G2, diluted 1:100, Novus Biologicals) for flavivirus-infected cells and murine-produced Clone 9E10 primary antibody specific for NS5A (kindly provided by Charles Rice at The Rockefeller University) diluted 1:8000 in FACS buffer (1% FBS (v/v) in DPBS) for HCV , .

Techniques: Infection, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Highly multiplexed mRNA quantitation with CRISPR-Cas13

doi: 10.1101/2023.08.16.553527

Figure Lengend Snippet: a , Percent of antigen-positive cells for HCV (2 MOIs). b , Percent of antigen-positive cells for flavivirus-infected cells. Error bars presented as SD. c , Infected cell populations for HCV samples. d , Infected cell populations for flavivirus samples.

Article Snippet: Pellets were subsequently incubated for one hour at room temperature with flavivirus group antigen antibody (clone 4G2, diluted 1:100, Novus Biologicals) for flavivirus-infected cells and murine-produced Clone 9E10 primary antibody specific for NS5A (kindly provided by Charles Rice at The Rockefeller University) diluted 1:8000 in FACS buffer (1% FBS (v/v) in DPBS) for HCV , .

Techniques: Infection