#METABOLOMICS WORKBENCH engelmab_20210112_011037 DATATRACK_ID:2394 STUDY_ID:ST001653 ANALYSIS_ID:AN002700 PROJECT_ID:PR001059 VERSION 1 CREATED_ON January 19, 2021, 4:18 pm #PROJECT PR:PROJECT_TITLE Untargeted metabolomics analysis of Mucosal-Associated-Invariant-T (MAIT) cells PR:PROJECT_TITLE stimulated with IL-12/IL-12, anti-CD3/CD28 or both PR:PROJECT_TYPE Untargeted metabolomics PR:PROJECT_SUMMARY MAIT cells are unique for their ability to recognize bacterial metabolites, PR:PROJECT_SUMMARY inducing an antigen(ag)-dependent activation, but can also be activated in an PR:PROJECT_SUMMARY ag-independent manner but the molecular details of MAIT cell activation are not PR:PROJECT_SUMMARY completely understood. In order to define the activation of MAIT cells on the PR:PROJECT_SUMMARY molecular level, among other things we applied untargeted metabolomics. PR:INSTITUTE Helmholtz Centre for Environmental Research PR:DEPARTMENT Molecular Systems Biology PR:LAST_NAME Engelmann PR:FIRST_NAME Beatrice PR:ADDRESS Permoserstraße 15, Leipzig, Saxony, 03418, Germany PR:EMAIL beatrice.engelmann@ufz.de PR:PHONE 00493412351099 #STUDY ST:STUDY_TITLE Mucosal-Associated-Invariant-T (MAIT) cells stimulated with IL-12/IL-12, ST:STUDY_TITLE anti-CD3/CD28 or both for 16 hours ST:STUDY_SUMMARY The untargeted metabolomics analysis was performed after metabolite extraction ST:STUDY_SUMMARY from vital cells. The main object of the study was to define the activation of ST:STUDY_SUMMARY MAIT cells on the molecular level. ST:INSTITUTE Helmholtz Centre for Environmental Research ST:LAST_NAME Engelmann ST:FIRST_NAME Beatrice ST:ADDRESS Permoserstraße 15, Leipzig, Saxony, 03418, Germany ST:EMAIL beatrice.engelmann@ufz.de ST:PHONE 00493412351099 ST:NUM_GROUPS 4 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS CD161+ TCR V 7.2+ MAIT cells were obtained by positive magnetic separation SU:CELL_STRAIN_DETAILS after isolation of Peripheral blood mononuclear cells from male human donors. #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Spender42_unstim_p Treatment:unstimulated RAW_FILE_NAME=Spender42_unstim_p.d SUBJECT_SAMPLE_FACTORS - Spender42_IL12_p Treatment:IL12/IL18 activation RAW_FILE_NAME=Spender42_IL12_p.d SUBJECT_SAMPLE_FACTORS - Spender42_CD3_p Treatment:a-CD3/a-CD28 activation RAW_FILE_NAME=Spender42_CD3_p.d SUBJECT_SAMPLE_FACTORS - Spender42_combi_p Treatment:combinated activation RAW_FILE_NAME=Spender42_combi_p.d SUBJECT_SAMPLE_FACTORS - Spender49_unstim_p Treatment:unstimulated RAW_FILE_NAME=Spender49_unstim_p.d SUBJECT_SAMPLE_FACTORS - Spender49_IL12_p Treatment:IL12/IL18 activation RAW_FILE_NAME=Spender49_IL12_p.d SUBJECT_SAMPLE_FACTORS - Spender49_CD3_p Treatment:a-CD3/a-CD28 activation RAW_FILE_NAME=Spender49_CD3_p.d SUBJECT_SAMPLE_FACTORS - Spender49_combi_p Treatment:combinated activation RAW_FILE_NAME=Spender49_combi_p.d SUBJECT_SAMPLE_FACTORS - Spender53_unstim_p Treatment:unstimulated RAW_FILE_NAME=Spender53_unstim_p.d SUBJECT_SAMPLE_FACTORS - Spender53_IL12_p Treatment:IL12/IL18 activation RAW_FILE_NAME=Spender53_IL12_p.d SUBJECT_SAMPLE_FACTORS - Spender53_CD3_p Treatment:a-CD3/a-CD28 activation RAW_FILE_NAME=Spender53_CD3_p.d SUBJECT_SAMPLE_FACTORS - Spender53_combi_p Treatment:combinated activation RAW_FILE_NAME=Spender53_combi_p.d SUBJECT_SAMPLE_FACTORS - Spender59_unstim_p Treatment:unstimulated RAW_FILE_NAME=Spender59_unstim_p.d SUBJECT_SAMPLE_FACTORS - Spender59_IL12_p Treatment:IL12/IL18 activation RAW_FILE_NAME=Spender59_IL12_p.d SUBJECT_SAMPLE_FACTORS - Spender59_CD3_p Treatment:a-CD3/a-CD28 activation RAW_FILE_NAME=Spender59_CD3_p.d SUBJECT_SAMPLE_FACTORS - Spender59_combi_p Treatment:combinated activation RAW_FILE_NAME=Spender59_combi_p.d SUBJECT_SAMPLE_FACTORS - Spender60_unstim_p Treatment:unstimulated RAW_FILE_NAME=Spender60_unstim_p.d SUBJECT_SAMPLE_FACTORS - Spender60_IL12_p Treatment:IL12/IL18 activation RAW_FILE_NAME=Spender60_IL12_p.d SUBJECT_SAMPLE_FACTORS - Spender60_CD3_p Treatment:a-CD3/a-CD28 activation RAW_FILE_NAME=Spender60_CD3_p.d SUBJECT_SAMPLE_FACTORS - Spender60_combi_p Treatment:combinated activation RAW_FILE_NAME=Spender60_combi_p.d #COLLECTION CO:COLLECTION_SUMMARY Peripheral blood mononuclear cells were isolated from male human donors at age CO:COLLECTION_SUMMARY 20-50 by Ficoll-Paque™ density-gradient centrifugation. CD161+ TCR Valpha 7.2+ CO:COLLECTION_SUMMARY MAIT cells were obtained by positive magnetic separation after isolation of CO:COLLECTION_SUMMARY Peripheral blood mononuclear cells from male human donors. CO:SAMPLE_TYPE Blood (whole) #TREATMENT TR:TREATMENT_SUMMARY MAIT cells were stimulated with 50ng/ml IL-12 and 50ng/ml IL-18, 10µg/ml TR:TREATMENT_SUMMARY plate-bound anti-CD3 and 1µg/ml soluble anti-CD28 or a combination of both. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were quenched 3 times with 1 ml ice-cold 0.9% sodium chloride. After SP:SAMPLEPREP_SUMMARY removing the quenching solution, cells were resuspended in 100 µl ice-cold SP:SAMPLEPREP_SUMMARY acetonitrile followed by 100 µl ice-cold Milli-Q water. After vortexing for 1 SP:SAMPLEPREP_SUMMARY min, cells were centrifuged (14000 rpm, 4 °C, 10 min). Supernatants were SP:SAMPLEPREP_SUMMARY transferred to new tubes. Intracellular metabolites were extracted again with SP:SAMPLEPREP_SUMMARY 500 µl Methanol:ACN:Milli-Q water (2:3:1). After centrifugation (14000 rpm, 4 SP:SAMPLEPREP_SUMMARY ° C, 10 min) both supernatants were combined, evaporated to dryness and stored SP:SAMPLEPREP_SUMMARY at -80 °C until measurement. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) CH:FLOW_RATE 0.3 ml/min CH:COLUMN_TEMPERATURE 45 CH:SOLVENT_A 0.1% formic acid in water CH:SOLVENT_B 0.1% formic acid in 50:50 ACN:MeOH CH:SAMPLE_INJECTION 10 µl #ANALYSIS AN:ANALYSIS_TYPE MS AN:DATA_FORMAT .d #MS MS:INSTRUMENT_NAME Agilent 6540 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass Hunter software was used to obtain raw data.Eluted metabolites were MS:MS_COMMENTS measured with the QTOF operated in centroid mode. Full scan data was generated MS:MS_COMMENTS with a scan range of 60-1600 m/z in positive ionization mode. Out of the survey MS:MS_COMMENTS scan the 5 most abundant precursor ions with charge state = 1 were subjected to MS:MS_COMMENTS fragmentation. The dynamic exclusion time was set at 30 s. Peak picking and MS:MS_COMMENTS database search was done using Progenesis QI software. MS:MS_RESULTS_FILE ST001653_AN002700_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END