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

Journal: PLoS ONE

Article Title: Comparative Immunogenicity of HIV-1 gp160, gp140 and gp120 Expressed by Live Attenuated Newcastle Disease Virus Vector

doi: 10.1371/journal.pone.0078521

Figure Lengend Snippet: Twenty-four h post-infection, the infected cells were fixed with paraformadehyde and permeabilized with Triton X-100 for detection of total antigen inside the cell. The cells were probed with a pool of gp120-specific monoclonal antibodies followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies, and analyzed by immunofluorescence. The cells were visualized under Nikon Eclipse TE fluorescent microscope. Arrows indicate areas of positive immunofluorescence.

Article Snippet: The plates were washed three times with plate washing solution (Synbiotics Carporation) and incubated for 1 h with a 1:1,000 dilution of an isotype-specific secondary antibody; namely, horseradish peroxidase (HRP)-conjugated goat anti-guinea pig IgG (KPL, Gaithersburg, MD), goat anti-guinea pig IgG1, goat anti-guinea pig IgG2a (Novus Biologicals, Littleton, CO), or sheep anti-guinea pig IgA (Immunology Consultants Laboratory, Newberg, OR).

Techniques: Infection, Bioprocessing, Incubation, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Comparative Immunogenicity of HIV-1 gp160, gp140 and gp120 Expressed by Live Attenuated Newcastle Disease Virus Vector

doi: 10.1371/journal.pone.0078521

Figure Lengend Snippet: The guinea pigs were immunized with the indicated rNDVs by the i.n. route. (A) The guinea pig sera were analyzed for NDV-specific antibodies by commercial NDV ELISA kits (Synbiotics Corporation). Mean ELISA end-point titers of NDV-specific serum antibodies on days 28 and 56 are shown. (B-D) The guine pig sera were analyzed by HIV-1 gp120 specific total IgG, IgG1 and IgG2 antibodies by isotype-specific ELISA with purified gp120. Mean ELISA end-point titers of gp120-binding serum antibodies of the indicated isotype on days 0, 7, 14, 21, 28, 42, 56, 70 and 90 are shown. Antibodies specific to gp120 were not detected in any animal on any day in the control rLaSota group. The graph shows the geometric mean value ± SEM for 3 animals in rLaSota, rLaSota/gp160 and rLaSota/gp140L groups, 6 animals in rLaSota/gp140S group and 5 animals in rLaSota/gp120 group. Arrows indicate time of rNDV immunizations on days 0 and 14. Statistical differences between the groups were calculated by unpaired t test (two-tailed). 1* indicates statistically significant differences ( P <0.05) of rLaSota/gp140S vs. rLaSota/gp160, rLaSota/gp140L and rLaSota/gp120 groups. 2* indicates statistically significant differences ( P <0.05) of rLaSota/gp140S vs. rLaSota/gp160 and rLaSota/gp140L groups.

Article Snippet: The plates were washed three times with plate washing solution (Synbiotics Carporation) and incubated for 1 h with a 1:1,000 dilution of an isotype-specific secondary antibody; namely, horseradish peroxidase (HRP)-conjugated goat anti-guinea pig IgG (KPL, Gaithersburg, MD), goat anti-guinea pig IgG1, goat anti-guinea pig IgG2a (Novus Biologicals, Littleton, CO), or sheep anti-guinea pig IgA (Immunology Consultants Laboratory, Newberg, OR).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Binding Assay, Control, Two Tailed Test

Journal: bioRxiv

Article Title: HNF1α transcriptional activation and repression maintain human islet α and β cell function

doi: 10.1101/2022.09.25.509394

Figure Lengend Snippet: RNA-seq of HNF1AKD β cells shows that HNF1α regulates insulin secretion, metabolism, developmental pathways, and cell-cell signaling in β cells. (A) Schematic of FACS scheme for isolation of transduced live β cells (HPi2+GFP+NTPDase3+) from control and HNF1AKD pseudoislets for downstream RNA sequencing. (B) HNF1A transcripts per million (TPM) in sequenced samples. (C) Differential expression (DE) analysis revealed significantly up- and down-regulated genes after HNF1AKD in β cells; thresholds: Fold change (FC)= 1.5, adjusted P -value= 0.05. (D) Heatmap of DEGs in β cells after HNF1AKD. Significantly (E) downregulated and (F) upregulated gene ontology (GO) pathways and (G) downregulated KEGG pathways in HNF1AKD relative to control β cells. P = Benjamini-Hochberg adjusted P -value; all P <0.05.

Article Snippet: Then, cells were stained with the following primary-secondary conjugated antibodies: HPi2-PE/Cy7 (Novus Biologicals NBP1-18946PECY7), NTPDase3-647 (clone hN3-B3S; Jean Sévigny Lab) and CD26-PE (BioLegend 302706).

Techniques: RNA Sequencing, Isolation, Control, Quantitative Proteomics

Journal: bioRxiv

Article Title: HNF1α transcriptional activation and repression maintain human islet α and β cell function

doi: 10.1101/2022.09.25.509394

Figure Lengend Snippet: RNA-seq of HNF1AKD α cells identifies dysregulation of calcium channel complexes and ATPase-coupled transmembrane transport, as well as hormone secretion pathways shared with β cells. (A) Schematic of methods for isolation of transduced α cells (HPi2+GFP+CD26+) from control and HNF1AKD pseudoislets for downstream RNA sequencing; (i) brightfield and (ii) blue light (488nm) images of human HNF1AKD pseudoislets, scale bars=1000μm. (B) HNF1A transcripts per million (TPM) in sequenced α cell samples. (C) Differential expression (DE) analysis revealed significantly up- and down-regulated genes after HNF1AKD in α cells; thresholds: FC= 1.5, adjusted P -value= 0.05. (D) Venn diagram comparing α versus β cell DEGs revealed shared and cell-specific consequences of HNF1AKD. Gene ontology (GO) pathways of (E) shared and (F) α cell enriched DEG sets. (G) Boxplots displaying TPM of select DEGs. P = Benjamini-Hochberg adjusted P -value; * P <0.05; all P <0.05 for GO pathways.

Article Snippet: Then, cells were stained with the following primary-secondary conjugated antibodies: HPi2-PE/Cy7 (Novus Biologicals NBP1-18946PECY7), NTPDase3-647 (clone hN3-B3S; Jean Sévigny Lab) and CD26-PE (BioLegend 302706).

Techniques: RNA Sequencing, Isolation, Control, Quantitative Proteomics

Journal: bioRxiv

Article Title: ARG1 could be expressed by human, but not represent for repair

doi: 10.1101/2020.07.02.183624

Figure Lengend Snippet: (A) The schematic diagram shows the time point after the pre-operation of GBR when tissues were harvested and the assays performed. (B and C) Analysis of RNA-seq data: both fpkm (B) and gene count (C) confirmed that ARG1 was not detected in the normal and inflammatory states, and its expression was low at the healing site, even when compared with other genes related to macrophages. (D) qPCR assay of ARG1 showed no significant differences between tissues from inflammatory and healing sites. Tissues were collected and digested to obtain single-cell suspensions for flow cytometry analysis. CD68and CD11b were used as markers for myeloid macrophages. iNOS and ARG1 were used as markers for M1 and M2 macrophages. (E) ARG1 expression was not detected in either group, and CD11b + CD68 + ARG1 + iNOS − macrophages were not observed using gated and t-distributed stochastic neighbor embedding (t-SNE) dimension reduction to visualize high-dimensional data. (F) Immunofluorescent staining did not reveal ARG1 in any group. Hoechst staining appears as blue in color while ARG1 appears as red. (G) Detection of surface markers by flow cytometry analysis. High expression of CD68 (CD68-FITC, Clone Y1/82A) and low expression of Arg1 were evident in the three groups. The population size of CD11b + CD68 + ARG1 − iNOS + macrophages was very small. Abbreviations: NB, normal alveolar bone tissue; IP, inflammatory periodontal tissue; H, healing (tissue harvest after guided bone regeneration surgery), and N/A, undetected.

Article Snippet: Cells were isolated by trypsinization after cultured for 1 (THP-1 monocytes to macrophages) and 7 (THP-1 monocytes derived macrophages after culturing in different circumstances) days respectively, co-incubated with antibodies against iNOS (iNOS-PE, Clone 4E5, Novus Biologicals, USA), CD68 (CD68-FITC, Clone Y1/82A, Abcam, UK), CD11b (PerCP/Cy5.5®, Clone M1/70, Abcam, UK) and ARG1 (ARG1-PE/Cyanine, Clone 14D2C43, BioLegend, USA) at 1:400 dilution in the dark for 1 h at 4°C (100μl per antibody for each sample).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: ARG1 could be expressed by human, but not represent for repair

doi: 10.1101/2020.07.02.183624

Figure Lengend Snippet: (A) Schematic diagram of the cell culture process and the time points for stimulation of macrophage differentiation, seeding macrophages in the different conditions, and performing the assays. (B) Light microscopy images of macrophages cultured under different conditions on Day 7. (C) Overlay of tSNE dimensionality reduction diagrams revealed the absence of AGR1 + macrophages in the flow cytometry analysis. (D) qPCR assay of ARG1 using Gapdh as the reference gene did not detect ARG1 in the four groups. (E-G) In contrast to the large population of CD11b + CD68 + macrophages (E) and CD68 + CD11b + iNOS + ARG − (F), CD68 + CD11b + iNOS − ARG1 + macrophages were not detected (G). Abbreviations: Ctrl, RPMI supplemented with 10% FBS; LPS, RPMI supplemented with 10% FBS and 10 ng/ml LPS; IL4, RPMI supplemented with 10% FBS, 10 ng/ml IL4; DBBM, RPMI supplemented with 10% FBS and 0.03 g/well DBBM; NS, no significance; N/A, undetected. *** denotes P < 0.001.

Article Snippet: Cells were isolated by trypsinization after cultured for 1 (THP-1 monocytes to macrophages) and 7 (THP-1 monocytes derived macrophages after culturing in different circumstances) days respectively, co-incubated with antibodies against iNOS (iNOS-PE, Clone 4E5, Novus Biologicals, USA), CD68 (CD68-FITC, Clone Y1/82A, Abcam, UK), CD11b (PerCP/Cy5.5®, Clone M1/70, Abcam, UK) and ARG1 (ARG1-PE/Cyanine, Clone 14D2C43, BioLegend, USA) at 1:400 dilution in the dark for 1 h at 4°C (100μl per antibody for each sample).

Techniques: Cell Culture, Light Microscopy, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Extensive Histopathological Characterization of Inflamed Bowel in the Dextran Sulfate Sodium Mouse Model with Emphasis on Clinically Relevant Biomarkers and Targets for Drug Development

doi: 10.3390/ijms22042028

Figure Lengend Snippet: List of antibodies used in the study.

Article Snippet: anti-IL-17A , Rat monoclonal, clone TC11-18H10; Novus Biologicals, Littleton, CO, USA , 1:50 , EDTA–Citrate pH 7.8.

Techniques: