1 / 6

Exploration of New Cell Lines: Characterization and Antibody Use in Macrophage Studies

This work focuses on the evaluation of seven cell lines, including J774A.1 and Raw 264.7, to explore their responsiveness to various stimuli. High ectopic expression of proteins was achieved using lentiviral methods, while RNA interfering showed effectiveness in macrophage lines. Preliminary assessments of AfCS assays have confirmed second messenger responses. Current antibody cocktails are prepared for macrophage use, and no issues with lipid analysis are anticipated. This study emphasizes improving throughput and fidelity of primary cell isolation compared to cultured cells.

garin
Télécharger la présentation

Exploration of New Cell Lines: Characterization and Antibody Use in Macrophage Studies

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. SummaryTesting New Cell Lines • Currently underway: exploration of 7 cell lines • (J774A.1, Raw 264.7, AtT-20, 3T3-L1, IC21, • Neuro 2a, N1E-115) • “A” list: J774A.1, Raw 264.7, AtT-20 • Rich responses (inputs and outputs) • Ectopic expression of proteins • 80-90% by lentivirus in Macrophage lines • 20-50% by transfection (Raw 264.7 cells, J774A.1 poorer) • RNAi works (in macrophage lines)

  2. SummaryTesting New Cell Lines • Preliminary assessment of AfCS assays • Second messenger responses (Ca, cAMP) verified • Current antibody cocktails are ready for use in macrophage lines • Macrophage lines suitable for microscopy • Anticipate no problems with lipid analysis • MA analysis should not be problematic • Protein trap – Expression levels higher than WEHI cells

  3. SummaryTransition from Current Focus • Higher throughput anticipated • Cultured cells vs isolation of primary cells from mice • Delays • Characterization of cell line stability • New libraries for Yeast 2 hybrid • Development of new assays • (chemotaxis, phagocytosis, secretion, etc.) • Data handling/display (transparent) • Ligand list

  4. Ligand List RAW264.7 (Literature) Other responses TGF-b c-Fms (M-CSF-R) IL-1b TNF IL6 Il-10 RANKL BNP IFN-g Fcg cAMP responses Ter (b-adrenergic) PGE2 (EP2, EP3, and EP4) VIP PACAP Ca responses MCP-1 (CCR2) C5a (C5a-R) UTP LPA Estrogen/Androgen

  5. GPCR List RAW264.7 (MA analysis) Adenosine A2b Adrenomedulin GRPbombesin Cannabinoid 1 Chemokine C-C 2 (Cmkbr2) Chemokine CCX CKR Chemokine C-X-C 3 (Cmkar3) Chemokine C-X3-C R1 (similar to) Cysteinyl leukotriene 1 Dopamine 3 Endothelin B Estrogen LPA (vzg-1) LPA 2 Malanocortin 5 Nerotensin 2 (B) NPY –Y1 PACAP 1 PGE 1 PGE EP2 P2Y 4 P2Y5 P2Y 12 PGE 4 5-HT 1B 5-HT 2C Somatostatin 2 SDF1 Stromal Cell Derived factor 2 V1a arginine vasopressin

  6. Ligand List for Cell Lines on “A List” Pathway Gs Gi Gq Other GPCR Other Rec. Toll-like, Cytokine, Tyr Kinase, Adh, other Total Raw 264.7 5 4 3 3 16 31 J774A.1 3 5 4 2 12 26 AtT-20 4 4 1 2 10 21

More Related