Journal: Cell Reports Medicine

Article Title: Circulating cancer-specific CD8 T cell frequency is associated with response to PD-1 blockade in Merkel cell carcinoma

doi: 10.1016/j.xcrm.2024.101412

Figure Lengend Snippet:

Article Snippet: TCF7 Antibody, clone C63D9, fluorophore PE-Cy7 , Cell Signaling Technology , Cat# 90511S; RRID: AB_3086656.

Techniques: Recombinant, Staining, Multiplex Assay, Software

Journal: eLife

Article Title: Hepatic lipid overload triggers biliary epithelial cell activation via E2Fs

doi: 10.7554/eLife.81926

Figure Lengend Snippet: ( A ) Scheme depicting bovine serum albumin (BSA) or fatty acid (FA) mix treatment for already grown BEC-organoids for 4 days. ( B ) Representative bright field and immunofluorescence (IF) images of lipids (BODIPY) and ACTIN co-staining, relative to panel A. n=4. ( C ) Measurement of triglyceride concentration upon FA mix treatment relative to panel A, normalized by protein amount. n=4. ( D ) Cell-titer Glo measurement for viability detection relative to panel A. n=4. ( E ) Representative hematoxylin and eosin (H&E) (top) and picrosirius red (bottom) staining of liver sections from C57BL/6 J mice fed chow diet (CD) or high-fat diet (HFD) for 15 weeks. The bar graph represents the body weight of the mice at the end of the experiment. n=10. ( F ) Representative Oil Red O staining, relative to panel E. n=8. ( G–H ) Representative cleaved caspase three staining ( G ) and quantification of apoptotic cells in bile ducts ( H ), relative to panel E. n=3. ( I ) Representative PANCK staining (left) and magnification (right), relative to panel E. n=5. ( L ) Flow cytometry gating strategy for analysis of BECs: individual cells were sequentially gated based on cell size (FSC-A versus SSC-A) and singlets. BECs were then selected based on EPCAM positivity after excluding leukocytes (CD45 + ), myeloid cells (CD11b + ), and endothelial cells (CD31 + ), yielding a population of single CD45 - /CD11b - / CD31 - /EPCAM + BECs. Data are shown as mean ± SEM. Absence of stars or ns, not significant (p>0.05); *p<0.05; ***p<0.001; one-way ANOVA with Dunnet’s test ( C, D ) or unpaired, two-tailed Student’s t-test ( E ) were used. PV, portal vein. Arrowheads mark bile ducts. Scale bars, 100 μm ( B, E, I - zoom out), 50 μm ( G - IF), and 20 μm ( F, G - bright field, I - zoom in).

Article Snippet: For flow cytometry analysis, livers were dissociated as described above, and single cells were stained for 20 min with BODIPY 558/568 (Invitrogen, D38D35) on ice, followed by incubation with fluorophore-conjugated antibodies CD45–PE/Cy7 (BD Biosciences, 552848), CD11b–PE/Cy7 (BD Biosciences, 552850), CD31–PE/Cy7 (BD Biosciences, 561410), EPCAM–VioBlue (Miltenyi Biotec, 130-123-871), and EdU Alexa Fluor 488 (ThermoFisher, C10425) for 30 min on ice.

Techniques: Immunofluorescence, Staining, Concentration Assay, Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: Hepatic lipid overload triggers biliary epithelial cell activation via E2Fs

doi: 10.7554/eLife.81926

Figure Lengend Snippet: ( A ) Fluorescence-activated cell sorting (FACS) gating strategy for isolation of CD45 − /CD11b − /CD31 − /EPCAM + BECs: individual cells were sequentially gated based on cell size (FSC-A versus SSC-A) and singlets. BECs were then selected based on EPCAM positivity after excluding leukocytes (CD45 + ), myeloid cells (CD11b + ), and endothelial cells (CD31 + ), yielding a population of single CD45 - /CD11b - / CD31 - /EPCAM + BECs. ( B ) Principal component analysis (PCA) of mRNAs measured in EPCAM + BECs from livers of mice fed chow diet (CD) or high-fat diet (HFD) by RNA-seq. n=5 for CD, n=7 for HFD. ( C ) Volcano plot of HFD vs. CD differential analysis. Top 20 differentially expressed genes were labeled. Blue dots represent downregulated genes (log2(FC) < –1 & adj. p-value <0.05). Red dots represent upregulated genes (log2(FC) >1 & adj. p-value <0.05). Gray dots represent genes not changing significantly. ( D–E ) Box plots representing the differential gene expression of Ncam1 ( D ) and well-established markers of the DR signature ( E ). n=5 for CD, n=7 for HFD. The Y-axis depicts log2(cpm +1) values. ( F ) Gene set enrichment analysis (GSEA) of KEGG terms. Top 15 enriched pathways (sorted by q-value). q-value: false discovery rate adjusted p-values. NES: normalized enrichment score. Data are summarized in boxplots. Absence of stars or ns, not significant (p>0.05); **p<0.01; unpaired, two-tailed Student’s t-test ( D, E ) was used.

Article Snippet: For flow cytometry analysis, livers were dissociated as described above, and single cells were stained for 20 min with BODIPY 558/568 (Invitrogen, D38D35) on ice, followed by incubation with fluorophore-conjugated antibodies CD45–PE/Cy7 (BD Biosciences, 552848), CD11b–PE/Cy7 (BD Biosciences, 552850), CD31–PE/Cy7 (BD Biosciences, 561410), EPCAM–VioBlue (Miltenyi Biotec, 130-123-871), and EdU Alexa Fluor 488 (ThermoFisher, C10425) for 30 min on ice.

Techniques: Fluorescence, FACS, Isolation, RNA Sequencing Assay, Labeling, Expressing, Two Tailed Test

Journal: eLife

Article Title: Hepatic lipid overload triggers biliary epithelial cell activation via E2Fs

doi: 10.7554/eLife.81926

Figure Lengend Snippet:

Article Snippet: For flow cytometry analysis, livers were dissociated as described above, and single cells were stained for 20 min with BODIPY 558/568 (Invitrogen, D38D35) on ice, followed by incubation with fluorophore-conjugated antibodies CD45–PE/Cy7 (BD Biosciences, 552848), CD11b–PE/Cy7 (BD Biosciences, 552850), CD31–PE/Cy7 (BD Biosciences, 561410), EPCAM–VioBlue (Miltenyi Biotec, 130-123-871), and EdU Alexa Fluor 488 (ThermoFisher, C10425) for 30 min on ice.

Techniques: Flow Cytometry, Viability Assay, Isolation, SYBR Green Assay, Activity Assay, Recombinant, Software, Immunohistochemistry, Immunofluorescence

Journal: eLife

Article Title: Hepatic lipid overload triggers biliary epithelial cell activation via E2Fs

doi: 10.7554/eLife.81926

Figure Lengend Snippet: ( A ) Scheme depicting bovine serum albumin (BSA) or fatty acid (FA) mix treatment for already grown BEC-organoids for 4 days. ( B ) Representative bright field and immunofluorescence (IF) images of lipids (BODIPY) and ACTIN co-staining, relative to panel A. n=4. ( C ) Measurement of triglyceride concentration upon FA mix treatment relative to panel A, normalized by protein amount. n=4. ( D ) Cell-titer Glo measurement for viability detection relative to panel A. n=4. ( E ) Representative hematoxylin and eosin (H&E) (top) and picrosirius red (bottom) staining of liver sections from C57BL/6 J mice fed chow diet (CD) or high-fat diet (HFD) for 15 weeks. The bar graph represents the body weight of the mice at the end of the experiment. n=10. ( F ) Representative Oil Red O staining, relative to panel E. n=8. ( G–H ) Representative cleaved caspase three staining ( G ) and quantification of apoptotic cells in bile ducts ( H ), relative to panel E. n=3. ( I ) Representative PANCK staining (left) and magnification (right), relative to panel E. n=5. ( L ) Flow cytometry gating strategy for analysis of BECs: individual cells were sequentially gated based on cell size (FSC-A versus SSC-A) and singlets. BECs were then selected based on EPCAM positivity after excluding leukocytes (CD45 + ), myeloid cells (CD11b + ), and endothelial cells (CD31 + ), yielding a population of single CD45 - /CD11b - / CD31 - /EPCAM + BECs. Data are shown as mean ± SEM. Absence of stars or ns, not significant (p>0.05); *p<0.05; ***p<0.001; one-way ANOVA with Dunnet’s test ( C, D ) or unpaired, two-tailed Student’s t-test ( E ) were used. PV, portal vein. Arrowheads mark bile ducts. Scale bars, 100 μm ( B, E, I - zoom out), 50 μm ( G - IF), and 20 μm ( F, G - bright field, I - zoom in).

Article Snippet: For FACS analysis, single cells were filtered with a 40 µm cell strainer (Falcon, 352340) and incubated with fluorophore-conjugated antibodies CD45–PE/Cy7 (BD Biosciences, 552848), CD11b–PE/Cy7 (BD Biosciences, 552850), CD31–PE/Cy7 (Abcam, ab46733), CD31-PE/Cy7 (BD Biosciences, 561410) and EPCAM–APC (eBioscience, 17-5791-82) for 30 min on ice.

Techniques: Immunofluorescence, Staining, Concentration Assay, Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: Hepatic lipid overload triggers biliary epithelial cell activation via E2Fs

doi: 10.7554/eLife.81926

Figure Lengend Snippet: ( A ) Fluorescence-activated cell sorting (FACS) gating strategy for isolation of CD45 − /CD11b − /CD31 − /EPCAM + BECs: individual cells were sequentially gated based on cell size (FSC-A versus SSC-A) and singlets. BECs were then selected based on EPCAM positivity after excluding leukocytes (CD45 + ), myeloid cells (CD11b + ), and endothelial cells (CD31 + ), yielding a population of single CD45 - /CD11b - / CD31 - /EPCAM + BECs. ( B ) Principal component analysis (PCA) of mRNAs measured in EPCAM + BECs from livers of mice fed chow diet (CD) or high-fat diet (HFD) by RNA-seq. n=5 for CD, n=7 for HFD. ( C ) Volcano plot of HFD vs. CD differential analysis. Top 20 differentially expressed genes were labeled. Blue dots represent downregulated genes (log2(FC) < –1 & adj. p-value <0.05). Red dots represent upregulated genes (log2(FC) >1 & adj. p-value <0.05). Gray dots represent genes not changing significantly. ( D–E ) Box plots representing the differential gene expression of Ncam1 ( D ) and well-established markers of the DR signature ( E ). n=5 for CD, n=7 for HFD. The Y-axis depicts log2(cpm +1) values. ( F ) Gene set enrichment analysis (GSEA) of KEGG terms. Top 15 enriched pathways (sorted by q-value). q-value: false discovery rate adjusted p-values. NES: normalized enrichment score. Data are summarized in boxplots. Absence of stars or ns, not significant (p>0.05); **p<0.01; unpaired, two-tailed Student’s t-test ( D, E ) was used.

Article Snippet: For FACS analysis, single cells were filtered with a 40 µm cell strainer (Falcon, 352340) and incubated with fluorophore-conjugated antibodies CD45–PE/Cy7 (BD Biosciences, 552848), CD11b–PE/Cy7 (BD Biosciences, 552850), CD31–PE/Cy7 (Abcam, ab46733), CD31-PE/Cy7 (BD Biosciences, 561410) and EPCAM–APC (eBioscience, 17-5791-82) for 30 min on ice.

Techniques: Fluorescence, FACS, Isolation, RNA Sequencing Assay, Labeling, Expressing, Two Tailed Test

Journal: eLife

Article Title: Hepatic lipid overload triggers biliary epithelial cell activation via E2Fs

doi: 10.7554/eLife.81926

Figure Lengend Snippet:

Article Snippet: For FACS analysis, single cells were filtered with a 40 µm cell strainer (Falcon, 352340) and incubated with fluorophore-conjugated antibodies CD45–PE/Cy7 (BD Biosciences, 552848), CD11b–PE/Cy7 (BD Biosciences, 552850), CD31–PE/Cy7 (Abcam, ab46733), CD31-PE/Cy7 (BD Biosciences, 561410) and EPCAM–APC (eBioscience, 17-5791-82) for 30 min on ice.

Techniques: Flow Cytometry, Viability Assay, Isolation, SYBR Green Assay, Activity Assay, Recombinant, Software, Immunohistochemistry, Immunofluorescence

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Chemokine Receptor CCR2 is Critical for Monocyte Accumulation and Survival in West Nile Virus Encephalitis

doi: 10.4049/jimmunol.1003003

Figure Lengend Snippet: WNV induction of monocyte accumulation in the CNS is selectively and strongly reduced in the absence of Ccr2: FACS analysis. Brain tissue was collected from uninfected Ccr2+/+ or Ccr2−/− mice (day 0) and mice at the indicated time points after infection with WNV. The total numbers of CD4+ T cells (A), CD8+ T cells (B), NK1.1+ cells (C), Ly6chiCD11b+ monocytes (D) and Ly6cintCD11b+ neutrophils (E) accumulating in the CNS were determined by flow cytometry. Representative FACS plots are shown for monocytes and neutrophils on day 11 (F). Data from 2 experiments were compiled and each data point represents the mean +/− SEM of 3 to 13 mice.

Article Snippet: Rat anti-mouse monoclonal antibodies coupled to the following fluorophores and directed against the following surface molecules were used for flow cytometry: FITC-conjugated CD19 (BD Biosciences) and Ly6c (eBiosciences); PE-conjugated CD115 (eBiosciences) and CD3 (BD Biosciences); PE-Cy-7-conjugated F4/80 (eBiosciences) and CD8α (eBiosciences); APC-conjugated CD45 (BD Biosciences) and NK1.1 (BD Biosciences); APC-Cy-7 conjugated CD11b (eBiosciences) and CD4 (eBiosciences).

Techniques: Infection, Flow Cytometry