Validation & Assay Performance Summary

Transcription

1 Validation & Assay Performance Summary CellSensor NFκB RAW. Cell Line Cat. no. K CellSensor Cell-Based Assay Validation Packet This cell-based assay has been thoroughly tested and validated by Invitrogen and is suitable for immediate use in a screening application. The following information illustrates the high level of assay testing completed and the validation of assay performance under optimized conditions. Pathway Description The molecules responsible for coordinating the immune system s recognition of pathogens are tolllike receptors (TLRs) of which there are currently identified families in humans. TLRs are pattern recognition receptors (PRRs), binding to pathogenassociated molecular patterns (PAMPs), small molecular sequences consistently found on pathogens. This binding activates the transcription of immune genes and regulators such as cytokines, chemokines, and co-stimulatory molecules, thereby promoting the initial immune response of macrophages and neutrophils. Cell Line Description The CellSensor NFκB-bla RAW. cell line contains a beta-lactamase reporter gene under control of the NFκB response element stably integrated into mouse monocyte RAW. cells. This cell line is a clonal population isolated in response Lipopolysaccharide (a TLR ligand) by flow cytometry. This cell line has also been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time and validated for Z and EC concentrations of TLR ligand: Imiquimod. Additional testing information using known inhibitors or activators of the pathway are also provided. Oct8

2 Validation Summary Testing and validation of this assay was evaluated in a 8-well format using LiveBLAzer -FRET B/G Substrate.. Primary agonist dose response under optimized conditions (n=) Imiquimod EC =. µg/ml Z -Factor (EC ) =. Response Ratio =. Optimum cell no. =, cells/well Optimum [DMSO] =.-.% Optimum Stim. Time = hrs Max. [Stimulation] = µg/ml Primary Agonist Dose Response Figure Imiquimod dose response under optimized conditions nm / nm UNSTIM STIM Day Day Day. Alternate Stimuli See Compound Panel Section. Small molecule inhibitor Testing See Compound Panel. Receptor Knockdown by Stealth RNAi. Cell culture and maintenance See Cell Culture and Maintenance Section and Table Assay Testing Summary. Assay performance with variable cell number. Assay performance with variable stimulation time NFκB-bla RAW. cells (, cells/well) were assayed on three separate days represented by the three curves shown on the graph. Cells were plated in a 8-well format and were stimulated with imiquimod (EMD Biosciences Cat.No. ) over the indicated concentration range in the presence of.% DMSO for hours. Cells were then loaded with LiveBLAzer -FRET B/G Substrate for hours. Fluorescence emission values at nm and nm were obtained using a standard fluorescence plate reader and the nm/nm ratio plotted for the indicated concentrations of imiquimod (n= for each data point). Images of unstimulated and stimulated cells are shown. Ligand Panel Figure NFκB-bla RAW. responds to ligands of various Toll-like Receptors 8. Assay performance with variable substrate loading time 9. Assay performance with variable DMSO concentration..... Unstim ng/ml LPS (TLR) µg/ml Poly I:C (TLR) µg/ml Imiquimod (TLR/8) µg/ml CpG Oligo (TLR9) Hour Hour µg/ml CpG neg. cntrl. NFκB-bla RAW. cells (, cells/well) were plated in a 8-well format in assay medium. They were treated for or hours with a panel of ligands at the indicated concentrations, and then loaded with LiveBLAzer -FRET B/G Substrate for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader and the Response Ratios plotted for each treatment (n=8 for each data point). Oct8

3 Figure NFκB-bla RAW. response silenced by IRAK / Inhibitor IC =. µm log [IRAK / Inhibitor µm] - %inhibition RR % inhibition NFκB-bla RAW. cells (, cells/well) were plated the day prior to the assay in a 8-well format in assay medium They were treated with the indicated concentration of IRAK ¼ Inhibitor (Calbiochem, ) and then stimulated with imiquimod (EMD Biosciences Cat.No. )) at the EC 8 for hours. The cells were then loaded with LiveBLAzer -FRET B/G Substrate for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader and the Response Ratios plotted for each treatment (n= for each data point). Receptor Knockdown by Stealth RNAi either no oligo, BlockIT fluorescent transfection-efficiency indicator, one of three different GC-content negative controls (Lo, Med, and Hi), β-lactamase positive control, or with one of three Mus musculus TLR-specific Stealth RNAi oligos (Invitrogen, ). They were transfected using Lipofectamine ( and incubated for hours. They were then stimulated with imiquimod (EMD Biosciences Cat.No. )) at the EC 8 for hours. The cells were then loaded with LiveBLAzer -FRET B/G Substrate for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader and the % Inhibition plotted for each treatment (n= for each data point). Cell Culture and Maintenance Thaw cells in Growth Medium without Blasticidin and culture them in Growth Medium with Blasticidin. Pass or feed cells at least twice a week and maintain them in a C/% CO incubator. Maintain cells between and 8% confluence. Note: We recommend passing cells for three passages after thawing before using them in the beta-lactamase assay. For more detailed cell growth and maintenance directions, please refer to the customer protocol. Figure NFκB-bla RAW. response silenced by knocking down TLR expression with Stealth RNAi % 9% 8% % Inhibition % % % % % % % % No Oligo LO GC MED GC HI GC B-LAC TLR- TLR- TLR- NFκB-bla RAW. cells (, cells/well) were plated in a 9-well format in assay medium. They were transfected with Oct8

4 Table Cell Culture and Maintenance Component Growth Medium Assay Medium Freezing Medium DMEM w/ GlutaMAX 9% -- OptiMEM % Dialyzed FBS (do not substitute!) %.% HEPES mm mm NEAA. mm. mm Sodium Pyruvate -- mm Penicillin (antibiotic) U/ml U/ml Streptomycin (antibiotic) µg/ml µg/ml Blasticidin (antibiotic) µg/ml Recovery Cell Culture Freezing Medium % Assay Performance with Variable Cell Number Figure Imiquimod dose response with different plating cell numbers/well Assay Performance with Variable Stimulation Time Figure Imiquimod dose response with hour and hour stimulation times k k k k NFκB-bla RAW. cells were plated with indicated number of cells/well in a 8-well format in assay medium. They were stimulated with indicated concentration of imiquimod (EMD Biosciences Cat.No. ) for hours. Cells were then loaded with LiveBLAzer -FRET B/G Substrate for hours. Fluorescence emission values at nm and nm for the each cell number. (n=8 for each data point) Hour Hour NFκB-bla RAW. cells were plated at, cells/well in a 8-well format in assay medium. hour cells were immediately treated with imiquimod (EMD Biosciences Cat.No. ) for hours. hour cells were incubated for hours then stimulated with imiquimod for hours. Cells were then loaded with LiveBLAzer -FRET B/G Substrate for hours. Fluorescence emission values at nm and nm for the each cell number against the indicated concentrations of imiquimod (n=8 for each data point). Oct8

5 Assay Performance with Variable Substrate Loading Time Figure Imiquimod dose response with various substrate loading times hour hour hour hour NFκB-bla RAW. cells were plated at, cells/well in a 8-well format in assay medium. They were stimulated with indicated concentration of imiquimod (EMD Biosciences Cat.No. ) for hours. Cells were then loaded with LiveBLAzer -FRET B/G Substrate for indicated hours. Fluorescence emission values at nm and nm for the each cell number against the indicated concentrations of imiquimod (n=8 for each data point). Assay Performance with Variable DMSO Concentration Figure Imiquimod dose response with,.,. and % DMSO %.%.%.% NFκB-bla RAW. cells were plated at, cells/well in a 8-well format in assay medium. They were stimulated with imiquimod (EMD Biosciences Cat.No. ) in the presence of indicated amount of DMSO for hours. Cells were then loaded with LiveBLAzer -FRET B/G Substrate for hours. Fluorescence emission values at nm and nm were obtained using a standard fluorescence plate reader and the Response Ratios for each DMSO concentration were plotted against the indicated concentrations of imiquimod (n=8 for each data point). References Zuany-Amorim, C., Hastewell, J., Walker, C. Toll-like Receptors as Potential Therapeutic Targets for Multiple Diseases. Nature Reviews Drug Discovery., 9-8 (). Krieg, Arthur. CpG motifs: the active ingredient in bacterial extracts? Nature Medicine. 9, 8-8 (). Chow, Jesse et al. Toll-like Receptor- Mediates Lipopolysaccharide-induced Signal Transduction. J.of Biological Chemistry., 89-9 (999). Oct8