#METABOLOMICS WORKBENCH amitch_20151222_9535751_mwtab.txt DATATRACK_ID:470 efahy_20160104_115042 STUDY_ID:ST000307 ANALYSIS_ID:AN000486 PROJECT_ID:PR000247 VERSION 1 CREATED_ON January 4, 2016, 11:50 am #PROJECT PR:PROJECT_TITLE CRBN's role in T cell activation PR:PROJECT_SUMMARY Targeting CRBN to alter CD8 T cell metabolism PR:INSTITUTE Moffitt Cancer Center PR:DEPARTMENT Immunology PR:LAST_NAME Burnette PR:FIRST_NAME Pearlie K. PR:ADDRESS - PR:EMAIL pearlie.burnette@moffitt.org PR:PHONE - #STUDY ST:STUDY_TITLE Targeting CRBN to alter CD8 T cell metabolism ST:STUDY_TYPE T cell activation ST:STUDY_SUMMARY The purpose of this study was to evaluate the metabolic profile of different ST:STUDY_SUMMARY treatments of T-Cells among two separate mouse genotypes. ST:INSTITUTE University of Florida ST:DEPARTMENT SECIM ST:LABORATORY Moffitt Cancer Center - Immunology ST:LAST_NAME Swearingen ST:FIRST_NAME Rebecca ST:ADDRESS - ST:EMAIL rebecca.swearingen@moffitt.org ST:PHONE - ST:NUM_GROUPS 12 ST:TOTAL_SUBJECTS 60 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:ANIMAL_ANIMAL_SUPPLIER WT (CRBN +/+) mice are from Jackson labs and CRBN -/- mice have been bred at SU:ANIMAL_ANIMAL_SUPPLIER Moffitt Cancer Center SU:ANIMAL_HOUSING Standard bedding + cotton pads, hepa filtered air SU:ANIMAL_LIGHT_CYCLE Dark 6pm-6am SU:ANIMAL_FEED Feed from Harlan, not sterile SU:ANIMAL_WATER autoclaved H2O SU:ANIMAL_INCLUSION_CRITERIA Animals used in this experiment were sex-matched and age matched. The ages were SU:ANIMAL_INCLUSION_CRITERIA 8-12 weeks old in both sets of experiments #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS sbs_706 CD3 + CD28 KO1 042215 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_707 CD3 + CD28 KO2 042215 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_708 CD3 + CD28 KO3 042215 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_709 CD3 + CD28 KO4 042215 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_710 CD3 + CD28 KO5 042215 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_711 CD3 KO1 042215 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_712 CD3 KO2 042215 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_713 CD3 KO3 042215 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_714 CD3 KO4 042215 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_715 CD3 KO5 042215 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_716 Naive KO1 042215 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_717 Naive KO2 042215 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_718 Naive KO3 042215 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_719 Naive KO4 042215 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_720 Naive KO5 042215 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_721 CD3 + CD28 WT1 042215 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_722 CD3 + CD28 WT2 042215 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_723 CD3 + CD28 WT3 042215 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_724 CD3 + CD28 WT4 042215 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_725 CD3 + CD28 WT5 042215 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_726 CD3 WT1 042215 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_727 CD3 WT2 042215 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_728 CD3 WT3 042215 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_729 CD3 WT4 042215 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_730 CD3 WT5 042215 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_731 Naive WT 1 042215 Strain:CRBN +/+ | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_732 Naive WT 2 042215 Strain:CRBN +/+ | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_733 Naive WT 3 042215 Strain:CRBN +/+ | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_734 Naive WT 4 042215 Strain:CRBN +/+ | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_735 Naive WT 5 042215 Strain:CRBN +/+ | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_736 CD3 + CD28 KO1 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_737 CD3 + CD28 KO2 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_738 CD3 + CD28 KO3 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_739 CD3 + CD28 KO4 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_740 CD3 + CD28 KO5 Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_741 CD3 KO1 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_742 CD3 KO2 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_743 CD3 KO3 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_744 CD3 KO4 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_745 CD3 KO5 Strain:CRBN -/- | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_746 Naive KO1 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_747 Naive KO2 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_748 Naive KO3 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_749 Naive KO4 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_750 Naive KO5 Strain:CRBN -/- | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_751 CD3 + CD28 WT1 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_752 CD3 + CD28 WT2 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_753 CD3 + CD28 WT3 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_754 CD3 + CD28 WT4 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_755 CD3 + CD28 WT5 Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_756 CD3 WT1 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_757 CD3 WT2 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_758 CD3 WT3 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_759 CD3 WT4 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_760 CD3 WT5 Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Gender=M SUBJECT_SAMPLE_FACTORS sbs_761 Naive WT 1 Strain:CRBN +/+ | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_762 Naive WT 2 Strain:CRBN +/+ | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_763 Naive WT 3 Strain:CRBN +/+ | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_764 Naive WT 4 Strain:CRBN +/+ | Treatment:unstimulated Gender=M SUBJECT_SAMPLE_FACTORS sbs_765 Naive WT 5 Strain:CRBN +/+ | Treatment:unstimulated Gender=M #COLLECTION CO:COLLECTION_SUMMARY - CO:SAMPLE_TYPE cell CO:COLLECTION_METHOD centrifuged in ficoll-gradient to remove debris, then washed in PBS CO:COLLECTION_TIME 0 or 24 hours after stimulation CO:STORAGE_CONDITIONS -80°C CO:COLLECTION_VIALS 15mL conical tubes CO:STORAGE_VIALS microfuge tubes #TREATMENT TR:TREATMENT_SUMMARY - TR:ANIMAL_ANESTHESIA CO2 asphyxiation TR:ANIMAL_ACCLIMATION_DURATION Mice from Jackson are subjected to a week-long acclimation period in our TR:ANIMAL_ACCLIMATION_DURATION facility prior to experimental use. TR:ANIMAL_ENDP_TISSUE_COLL_LIST Spleen into cell culture media TR:CELL_GROWTH_CONTAINER 96-well round-bottom plate that was coated with anti-CD3 antibody TR:CELL_GROWTH_RATE Murine T cells generally divide after 48-72 hours of stimulation. TR:CELL_MEDIA RPMI 1640 with 10% FBS, pen/strep, gentamycin, NEAA, Sodium pyruvate, fungizone TR:CELL_MEDIA (Amphotericin B), 2-mercaptoethanol TR:CELL_ENVIR_COND 37°C, normoxic conditions #SAMPLEPREP SP:SAMPLEPREP_SUMMARY - SP:SAMPLEPREP_PROTOCOL_FILENAME Consolidated Cellular Metabolomics with DNA quant.pdf SP:SAMPLE_SPIKING Appendix A - Internal Standard Prep GLCMS.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Scientific-Dionex Ultimate 3000 CH:COLUMN_NAME ACE Excel 2 C18-PFP (100 x 2.1mm, 2um) CH:METHODS_FILENAME Metabolomics_LCMSProtocol.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Garrett Lab AN:ANALYSIS_PROTOCOL_FILE Metabolomics_LCMSProtocol.pdf #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples CD3 KO4 042215 CD3 KO3 Naive KO1 042215 CD3 WT2 Naive KO3 042215 Naive KO5 CD3 WT3 042215 Naive WT 1 CD3 + CD28 KO2 042215 Naive KO1 Naive WT 5 042215 CD3 + CD28 KO1 Naive KO4 042215 CD3 + CD28 KO2 CD3 + CD28 KO4 CD3 + CD28 KO5 CD3 KO5 042215 CD3 WT2 042215 CD3 KO4 CD3 WT1 CD3 + CD28 WT2 CD3 KO3 042215 CD3 + CD28 KO3 042215 CD3 + CD28 WT4 CD3 + CD28 KO3 CD3 WT4 042215 CD3 WT4 CD3 WT5 CD3 KO2 042215 CD3 WT5 042215 Naive WT 1 042215 Naive WT 4 042215 CD3 + CD28 KO5 042215 Naive WT 4 CD3 + CD28 WT5 042215 Naive WT 3 Naive WT 2 Naive KO2 Naive WT 2 042215 Naive KO4 CD3 WT3 CD3 + CD28 WT3 CD3 KO1 042215 CD3 + CD28 WT3 042215 Naive KO3 CD3 + CD28 WT2 042215 CD3 + CD28 KO4 042215 Naive KO5 042215 CD3 WT1 042215 Naive WT 3 042215 CD3 + CD28 WT1 042215 CD3 + CD28 WT5 CD3 + CD28 WT1 CD3 KO2 CD3 KO1 Naive WT 5 CD3 + CD28 WT4 042215 CD3 KO5 Naive KO2 042215 CD3 + CD28 KO1 042215 Factors Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN +/+ | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN +/+ | Treatment:unstimulated Strain:CRBN +/+ | Treatment:anti-CD3 & anti-CD28 stimulation Strain:CRBN -/- | Treatment:anti-CD3 stimulation Strain:CRBN -/- | Treatment:unstimulated Strain:CRBN -/- | Treatment:anti-CD3 & anti-CD28 stimulation MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index quant_moverz METABOLITES_END #END