nih 3t12 mouse fibroblast cells  (ATCC)

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: High-dimensional single-cell protein analysis of MHV68 infection by CyTOF. (A) 3T12 fibroblasts were either mock or MHV68-infected with WT MHV68.LANAβlac (MOI=0.5), with indicated populations FACS purified according to LANA::β-lactamase expression at 16 hours pi. (B) PhenoGraph analysis of mock, LANA- and LANA+ cells subjected to CyTOF analysis and visualized by tSNE-based dimensionality reduction identified 14 cell clusters (colored by cluster ID). (C) Comparison of the frequency (left) and protein expression profile (right) of PhenoGraph-defined clusters in LANA- and LANA+ samples. Clusters (in rows), ranked from those exclusively in LANA- samples (top) to those exclusively in LANA+ samples (bottom); asterisks indicate clusters statistically significantly different between conditions, defined by unpaired t-test corrected for multiple comparisons using the Holm-Sidak method, p<0.05. Data show mean ± SEM with individual symbols indicating individual sample values (n=4 per group). Relative protein expression for each cluster (right panel) is indicated by heatmap intensity, with protein markers divded by functional categories. (D) tSNE plot of LANA- and LANA+ cells colored according to LANA- and LANA+ exclusivity (left), vRCA expression (middle), and pH2AX expression (right panel). Red and blue lines identify events exclusive to LANA+ or LANA- clusters (left panel). Ruler defines range of expression with values calculated using the equation arcsinh (x/5) where x is raw expression value. Live, DNA+ cells ( 191 Ir+ 193 Ir+ 195 Pt-) were imported into PhenoGraph with 9,162 events total analyzed (1,018 events from each sample; n=1, mock, n=4 each for LANA+ and LANA- samples), clustered on 9 cell surface proteins (vRCA, BST2, CD9, CD29, CD44, CD63, Ly6A/E, Ly6C, and MHCI). See also Figure S1.

Article Snippet: NIH 3T12 cells (ATCC # CCL-164) were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco, Life Technologies) supplemented with 5% fetal bovine serum (Atlanta Biologicals), 1x Penicillin-Streptomycin-L-Glutamine (Gibco, Life Technologies).

Techniques: Infection, Purification, Expressing, Comparison, Functional Assay

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: Flow cytometric analysis of protein and RNA expression in 3T12 fibroblasts infected with MHV68 (MOI=0.5, harvested 16 hpi). (A) Analysis of vRCA and pH2AX protein expression in mock and MHV68-infected cultures identified four populations denoted by numbers and color (1=Gray, 2=Blue, 3=Orange, 4=Red). (B) Analysis of viral ORF18 and host Actin (Actb) mRNA expression across four populations of MHV68-infected cultures, stratified by vRCA and pH2AX expression. Border color of biaxial plots corresponds to the parent population stratified by protein expression in panel A. (C) Quantitaiton of the frequency of cells based on RNA expression profile as in panel B, depicting mean ± SEM from 3 independent experimental samples. Flow cytometric analysis was done on singlets, following gating to remove doublets.

Article Snippet: NIH 3T12 cells (ATCC # CCL-164) were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco, Life Technologies) supplemented with 5% fetal bovine serum (Atlanta Biologicals), 1x Penicillin-Streptomycin-L-Glutamine (Gibco, Life Technologies).

Techniques: RNA Expression, Infection, Expressing

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: scRNA-seq analysis of MHV68 viral gene expression in LANA+ 3T12 fibroblasts as in . (A) LANA+ cells show a wide range in how many viral genes are expressed on a per-cell basis. Expression of a viral gene was defined conservatively as any viral gene with ≥1 UMI detected per cell, with data showing the distribution of cells based on number of viral genes expressed per cell. (B) Viral genes vary widely in mean UMI per cell and the frequency of cells expressing an individual viral gene. Mean viral UMI per cell was calculated from positive cells that expressed ≥1 UMI per gene, with five representative genes identified by unique shading. Each dot represents expression for a single viral gene product for all 80 annotated MHV68 open reading frames. (C) The distribution of cells based on total viral UMIs reveals a bimodal distribution of virus low and virus high cells. (D) Total viral UMIs per cell as a function of the number of viral genes detected per cell (as in panel A). Cells demonstrate a bimodal distribution of total viral UMIs with virus high and low cells, with a positive Spearman correlation coefficient, rs, as indicated. (E) Frequency of cells expressing each viral gene, with viral gene expression defined as ≥1 UMI for each viral gene. Dashed line indicates 50% of events. (F) Mean UMI count per viral gene, defined by calculating mean value only from positive cells (≥1 UMI for each viral gene), depicting mean ± SD. Data represent scRNA-seq data from all LANA+ cells (n=1605 cells). nd, genes that were not detected. See also Figure S3 and Table S1.

Article Snippet: NIH 3T12 cells (ATCC # CCL-164) were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco, Life Technologies) supplemented with 5% fetal bovine serum (Atlanta Biologicals), 1x Penicillin-Streptomycin-L-Glutamine (Gibco, Life Technologies).

Techniques: Gene Expression, Expressing, Virus

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: scRNA-seq analysis of MHV68 gene expression in LANA+ 3T12 fibroblasts as depicted in . Comprehensive visualization of viral gene expression on a single cell basis. Each row depicts expression pattern for a different viral gene. Each column depicts expression within an individual cell. Cells are rank ordered from the cell with the least (left) to the greatest total viral UMI count (right), with corresponding total viral (filled orange line) and host (black dots) UMIs per cell depicted in the bottom panel. Cells are further bisected into Virus low cells (< 500 viral UMIs per cell) and Virus high cells (> 500 viral UMIs per cell) by a thick vertical black line, from a total of 1605 cells. UMI counts are stratified based on the indicated heatmap scale. See also Figure S4.

Article Snippet: NIH 3T12 cells (ATCC # CCL-164) were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco, Life Technologies) supplemented with 5% fetal bovine serum (Atlanta Biologicals), 1x Penicillin-Streptomycin-L-Glutamine (Gibco, Life Technologies).

Techniques: Gene Expression, Expressing, Virus