Journal: bioRxiv

Article Title: Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer

doi: 10.1101/2021.02.04.429827

Figure Lengend Snippet: (A) An illustration of the mesothelial cell/apCAF-T cell co-culture assay. Podoplanin + MHC II + mesothelial cells from normal pancreata (0.55%) or apCAFs from KPfC tumors (2.45%) were sorted by FACS. The sorted mesothelial cells or apCAFs were incubated with the OVA peptide (OVA 323-339) for 4 hrs. Cells were then washed and co-cultured with CD4 + T cells isolated from OT II mice for 18 hrs. A similar assay was also performed using PanMeso cells. PanMeso cells were pre-treated with control medium or PDA organoid conditioned medium for 48 hrs and washed before the incubation with OVA peptide and being co-cultured with CD4 + T cells. (B-F) The CD4 + T cells after being co-cultured with mesothelial cells/apCAFs (A) were subjected to flow cytometry for the analysis of the early activation markers of TCR ligation CD25 and CD69 (B and D), Treg markers CD25 and FoxP3 (C and E) as well as proliferation marker Ki67 (F). *P<0.05, ****P<0.0001 vs without OVA control. #### P<0.0001 vs normal mesothelial cells.

Article Snippet: The cell suspensions were then washed and stained with antibodies against CD4 (BD Biosciences, 1:100), CD25 (BD Biosciences, 1:100) and CD69 (BD Biosciences, 1:100) for 1 hr at 4°C.

Techniques: Co-culture Assay, Incubation, Cell Culture, Isolation, Flow Cytometry, Activation Assay, Ligation, Marker

Journal: bioRxiv

Article Title: Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer

doi: 10.1101/2021.02.04.429827

Figure Lengend Snippet: (A) The differential genes that were up-regulated in apCAFs compared to normal mesothelial cells from the integrated data were subjected to motif enrichment analysis. Top transcription factors from the analysis were shown. (B) The PanMeso cells were treated with control medium or PDA organoid conditioned medium for 48 hrs. Cell lysates were harvested and subjected to western blot, probing for the phosphorylation of the NF-κB signaling (P-P65) and TGFβ signaling (P-SMAD2) proteins. (C-D) The PanMeso cells were treated with IL-1α (5 ng/mL), TGFβ (30 ng/mL) or a combination of IL-1α (5 ng/mL) and TGFβ (30 ng/mL) for 48 hrs. Cells were then harvested and subjected to qPCR for mesothelial ( Msln , Upk3b , Ezr , Nkain4 , Cdh11 , Pdpn ) (C) and fibroblastic ( Il1a , Il6 , Cxcl1 , Cxcl12 , Col1a1 , Col1a2 , Col12a1 , Tgfb1 ) (D) genes. n=3, *P<0.05, **P<0.01, ***P<0.001. (E-H) The PanMeso cells were pre-treated with IL-1α (5 ng/mL), TGFβ (30 ng/mL) or a combination of IL-1α (5 ng/mL) and TGFβ (30 ng/mL) for 48 hrs and washed. Cells were then incubated with the OVA peptide (OVA 323-339) for 4 hrs. The PanMeso cells were washed and co-cultured with CD4 + T cells isolated from OT II mice for 18 hrs. The CD4 + T cells after the co-culture were subjected to flow cytometry for the analysis of the early activation markers of TCR ligation CD25 and CD69 (E and F) and the Treg markers CD25 and FoxP3 (G and H). ****P<0.0001 vs without OVA control. ## P<0.01, ### P<0.001, #### P<0.0001 vs control with OVA treatment.

Article Snippet: The cell suspensions were then washed and stained with antibodies against CD4 (BD Biosciences, 1:100), CD25 (BD Biosciences, 1:100) and CD69 (BD Biosciences, 1:100) for 1 hr at 4°C.

Techniques: Western Blot, Incubation, Cell Culture, Isolation, Co-Culture Assay, Flow Cytometry, Activation Assay, Ligation

Journal: Cell Reports Methods

Article Title: Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip

doi: 10.1016/j.crmeth.2022.100256

Figure Lengend Snippet:

Article Snippet: Mouse anti-CD69 V450 , BD Biosciences , Cat#560740; RRID: AB_1727512.

Techniques: Recombinant, Staining, Cell Isolation, Plasmid Preparation, Software