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)

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

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

Ligand List RAW264.7 (Literature)

cAMP responsesTer-adrenergic)PGE2 (EP2, EP3, and EP4)VIP PACAP

Ca responsesMCP-1 (CCR2) C5a (C5a-R)UTPLPAEstrogen/Androgen

Other responsesTGF-c-Fms (M-CSF-R) IL-1 TNFIL6Il-10RANKLBNPIFN-Fc

GPCR List RAW264.7 (MA analysis)

Adenosine A2bAdrenomedulin GRPbombesin Cannabinoid 1Chemokine C-C 2 (Cmkbr2)Chemokine CCX CKRChemokine C-X-C 3 (Cmkar3)Chemokine C-X3-C R1 (similar to)Cysteinyl leukotriene 1Dopamine 3Endothelin B Estrogen LPA (vzg-1)LPA 2Malanocortin 5

Nerotensin 2 (B)NPY –Y1PACAP 1PGE 1PGE EP2P2Y 4P2Y5P2Y 12PGE 45-HT 1B5-HT 2C Somatostatin 2SDF1Stromal Cell Derived factor 2V1a arginine vasopressin

Ligand List for Cell Lines on “A List”

Pathway Gs

Gi

Gq

Other GPCR

Other Rec. Toll-like, Cytokine, Tyr Kinase, Adh, otherTotal

Raw 264.75

4

3

3

16

31

J774A.13

5

4

2

12

26

AtT-204

4

1

2

10

21