Non-invasive High-Throughput Single-Cell Analysis of the Intracellular ph of Yeast by

Transcription

1 Non-invasive High-Throughput Single-Cell Analysis of the Intracellular of Yeast by Ratiometric Flow Cytometry Running title: Analysis of Yeast by Ratiometric Flow Cytometry Mari Valkonen 3, Dominik Mojzita 3, Merja Penttilä 3, Mojca Benčina 1,2# SUPPLEMENTAL MATERIAL 1

2 SUPPLEMENTAL TABLES Table S1: Nucleotide sequences of plasmids p-luorin-lexa-ad and pmv118 with brief description. p-luorin2-lexa-ad Plasmid p-luorin2-lexa-ad was constructed on the backbone of plasmid pyx22 (R&D Systems, Wiesbaden, Germany). It contains a luorin2 gene (PMID: , GeneArt/Invitrogen) under the control of the modified GAL1 promoter with 8 copies of a LexA operator (source plasmid psh18-34), as well as a fusion gene that consists of a LexA+SV4-NLS+B42-transcription activation domain under the control of the MET17 promoter. GACGTCCCCAGCTTGGAATTCGACAGGTTATCAGCAACAACACAGTCATATCCATTCTCAATTAGCTCTACCACAGTGTGTGAACCAATGTATCCAGCACCACCTGTAACCAAAACAATTTTAGAAGTAC TTTCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCCATATACATA TCCATATCTAATCTTACCTCGACTGCTGTATATAAAACCAGTGGTTATATGTACAGTACTGCTGTATATAAAACCAGTGGTTATATGTACAGTACGTCGACTGCTGTATATAAAACCAGTGGTTATATGT ACAGTACTGCTGTATATAAAACCAGTGGTTATATGTACAGTACGTCGAGGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGA TATATAAATGCAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTC AAGGAGAAAAAACTATAATGACTAAATCTCATTCAGAAGAAGTGATTGTACCTGAGTTCAATTCTAGCGCAAAGGAATTACCAAGACCATTGGCCGAAAAAATGTCTAAGGGTGAAGAATTATTCACTGG TGTTGTTCCAATCTTGGTTGAATTGGATGGTGATGTTAACGGTCACAAGTTTTCTGTTTCTGGTGAAGGTGAAGGTGATGCTACTTATGGTAAATTGACCTTGAAGTTCATCTGTACCACAGGTAAATTG CCAGTTCCATGGCCAACTTTGGTTACTACTTTGTCTTATGGTGTCCAATGCTTCTCTAGATACCCAGATCATATGAAGCAACACGACTTTTTCAAATCCGCTATGCCAGAAGGTTACGTTCAAGAAAGAA CCATCTTCTTCAAGGATGACGGTAACTACAAAACTAGAGCCGAAGTTAAGTTCGAAGGTGATACCTTGGTTAACAGAATCGAATTGAAGGGTATCGACTTCAAAGAAGATGGTAACATCTTGGGTCATAA GTTGGAATACAACTACAACGAACACTTGGTTTACATCATGGCCGATAAGCAAAAGAATGGTACTAAGGCCATTTTCCAAGTTCACCACAATATCGAAGATGGTAGTGTTCAATTGGCCGATCATTACCAA CAAAACACTCCAATTGGTGATGGTCCAGTTTTGTTGCCAGATAACCATTACTTGCATACCCAATCTGCTTTGTCTAAGGACCCAAACGAAAAAAGAGATCACATGGTCTTGTTGGAATTCGTTACTGCTG CTGGTATTACTCATGGTATGGACGAATTATACAAGTAATGACCGTCGACCTCGAGTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTA GACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAAC CTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCGGCCGGTACCCAACCGGATGCAAGGGTTCGAATCCCTTAGCTCTCATTATTTTTTGCTTTTTCTCTTGAGGTCACATGATCGCAAAA TGGCAAATGGCACGTGAAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGTTAGTCAAGGCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAAGTGTCGAAAAGGTGGCA CCTTGTCCAATTGAACACGCTCGATGAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTTTCCTTTTTCTTGCTCTCTTGTCTTTTCATCTACTATTTCCTTCGTGTAA TACAGGGTCGTCAGATACATAGATACAATTCTATTACCCCCATCCATACTCTAGACAATGAAAGCGTTAACGGCCAGGCAACAAGAGGTGTTTGATCTCATCCGTGATCACATCAGCCAGACAGGTATGC CGCCGACGCGTGCGGAAATCGCGCAGCGTTTGGGGTTCCGTTCCCCAAACGCGGCTGAAGAACATCTGAAGGCGCTGGCACGCAAAGGCGTTATTGAAATTGTTTCCGGCGCATCACGCGGGATTCGTCT GTTGCAGGAAGAGGAAGAAGGGTTGCCGCTGGTAGGTCGTGTGGCTGCCGGTGAACCACTTCTGGCGCAACAGCATATTGAAGGTCATTATCAGGTCGATCCTTCCTTATTCAAGCCGAATGCTGATTTC CTGCTGCGCGTCAGCGGGATGTCGATGAAAGATATCGGCATTATGGATGGTGACTTGCTGGCAGTGCATAAAACTCAGGATGTACGTAACGGTCAGGTCGTTGTCGCACGTATTGATGACGAAGTTACCG TTAAGCGCCTGAAAAAACAGGGCAATAAAGTCGAACTGTTGCCAGAAAATAGCGAGTTTAAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTTCACCATTGAAGGGCTGGCGGTTGGGGTTATTCGCAA CGGCGACTGGCTGGAATTCCCTCCAAAAAAGAAGAGAAAGGTAGCCCCTGCTGGTATCAATAAAGATATCGAGGAGTGCAATGCCATCATTGAGCAGTTTATCGACTACCTGCGCACCGGACAGGAGATG CCGATGGAAATGGCGGATCAGGCGATTAACGTGGTGCCGGGCATGACGCCGAAAACCATTCTTCACGCCGGGCCGCCGATCCAGCCTGACTGGCTGAAATCGAATGGTTTTCATGAAATTGAAGCGGATG TTAACGATACCAGCCTCTTGCTGAGTGGAGATGCCTCCTGATAGGGCATGGACGAGTTATACAAGTAACTCGAGCTCAGCTAGCTAACTGAATAAGGAACAATGAACGTTTTTCCTTTCTCTTGTTCCTA GTATTAATGACTGACCGATACATCCCTTTTTTTTTTTGTCTTTGTCTAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTCAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGT AGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTC AAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGGTTTACGGCACCTCGACCCCAAAACAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGA GCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAGATCCGTCGAGTTCAAGAGAAA AAAAAAGAAAAAGCAAAAAGAAAAAAGGAAAGCGCGCCTCGTTCAGAATGACACGTATAGAATGATGCATTACCTTGTCATCTTCAGTATCATACTGTTCGTATACATACTTACTGACATTCATAGGTAT ACATATATACACATGTATATATATCGTATGCTGCAGCTTTAAATAATCGGTGTCACTACATAAGAACACCTTTGGTGGAGGGAACATCGTTGGTTCCATTGGGCGAGGTGGCTTCTCTTATGGCAACCGC AAGAGCCTTGAACGCACTCTCACTACGGTGATGATCATTCTTGCCTCGCAGACAATCAACGTGGAGGGTAATTCTGCTTGCCTCTGCAAAACTTTCAAGAAAATGCGGGATCATCTCGCAAGAGAGATCT CCTACTTTCTCCCTTTGCAAACCAAGTTCGACAACTGCGTACGGCCTGTTCGAAAGATCTACCACCGCTCTGGAAAGTGCCTCATCCAAAGGCGCAAATCCTGATCCAAACCTTTTTACTCCACGCGCCA GTAGGGCCTCTTTAAATGCTTGACCGAGAGCAATCCCGCAGTCTTCAGTGGTGTGATGGTCGTCTATGTGTAAGTCACCAATGCACTCAACGATTAGCGACCAGCCGGAATGCTTGGCCAGAGCATGTAT CATATGGTCCAGAAACCCTATACCTGTGTGGACGTTAATCACTTGCGATTGTGTGGCCTGTTCTGCTACTGGTTCTGCCTCTTTTTCTGGGAAGATCGAGTGCTCTATCGCTAGGGGACCAGCCTTTAAA GAGATCGCAATCTGAATCTTGGTTTCATTTGTAATACGCTTTACTAGGGCTTTCTGCTCTGTCATCTTTGCCTTCGTTTATCTTGCCTGCTCATTTTTTAGTATATTCTTCGAAGAAATCACATTACTTT ATATAATGTATAATTCATTATGTGATAATGCCAATCGCTAAGAAAAAAAAAGAGTCATCCGCTAGGGGAAAAAAAAAAATGAAAATCATTACCGAGGCATAAAAAAATATAGAGTGTACTAGAGGAGGCC AAGAGTAATAGAAAAAGAAAATTGCGGGAAAGGACTGTGTTATGACTTCCCTGACTAATGCCGTGTTCAAACGATACCTGGCAGTGACTCCTAGCGCTCACCAAGCTCTTAAAACGGGAATTTATGGTGC ACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCG GGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTGCGGCCGCACTCT CACTAGTACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCGTGATAAATGCTTCAATAATATTGA AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTG CACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGA CGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGGACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAG CCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATC GTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATC TTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA GCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG TGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT GTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACA TGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCC AATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTACCTCACTCATTAGGCACCC CAGGCTTTACACTTTATGCTTCCGGCTCCTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATACGACTCACTATAGGGCGAATTG GGTACCGGGCCGGCCGTCGGGCCGTCGAGCTTGATGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGAAA AAAAGCGGTTAGCTCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGGAACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTG ACTGGTGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGT TCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAA GGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGATACATATTTGAATGTATT TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCT 2

3 pmv118 The luorin gene was cloned into vector B2158, a modified vector from pyx242 (R&D systems, UK) with a constitutive TPI1 promoter, resulting in plasmid pmv118. GATCTACGTATGGTCATTTCTTCTTCAGATTCCCTCATGGAGAAAGTGCGGCAGATGTATATGACAGAGTCGCCAGTTTCCAAGAGACTTTATTCAGGCACTTCCATGATAGGCAAGAGAGAAGACCCAG AGATGTTGTTGTCCTAGTTACACATGGTATTTATTCCAGAGTATTCCTGATGAAATGGTTTAGATGGACATACGAAGAGTTTGAATCGTTTACCAATGTTCCTAACGGGAGCGTAATGGTGATGGAACTG GACGAATCCATCAATAGATACGTCCTGAGGACCGTGCTACCCAAATGGACTGATTGTGAGGGAGACCTAACTACATAGTGTTTAAAGATTACGGATATTTAACTTACTTAGAATAATGCCATTTTTTTGA GTTATAATAATCCTACGTTAGTGTGAGCGGGATTTAAACTGTGAGGACCTTAATACATTCAGACACTTCTGCGGTATCACCCTACTTATTCCCTTCGAGATTATATCTAGGAACCCATCAGGTTGGTGGA AGATTACCCGTTCTAAGACTTTTCAGCTTCCTCTATTGATGTTACACCTGGACACCCCTTTTCTGGCATCCAGTTTTTAATCTTCAGTGGCATGTGAGATTCTCCGAAATTAATTAAAGCAATCACACAA TTCTCTCGGATACCACCTCGGTTGAAACTGACAGGTGGTTTGTTACGCATGCTAATGCAAAGGAGCCTATATACCTTTGGCTCGGCTGCTGTAACAGGGAATATAAAGGGCAGCATAATTTAGGAGTTTA GTGAACTTGCAACATTTACTATTTTCCCTTCTTACGTAAATATTTTTCTTTTTAATTCTAAATCAATCTTTTTCAATTTTTTGTTTGTATTCTTTTCTTGCTTAAATCTATAACTACAAAAAACACATAC AGGAATTCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAA ACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTC AAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTA TTGATTTTAAAGATGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACGAGCACTTGGTGTACATCATGGCAGACAAACAAAAGAATGGTACCAAAGCTATCTTTCAAGTTCACCACAACAT TGAAGATGGAGGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGCACACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAG AGAGACCACATGGTCTTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAGGATCCTCTAGAGTCGAGCTCAGCTAGCTAACTGAATAAGGAACAATGAACGTTT TTCCTTTCTCTTGTTCCTAGTATTAATGACTGACCGATACATCCCTTTTTTTTTTTGTCTTTGTCTAGCTCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTG ACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATG GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGT TTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGG TTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATCGACCGGTCG AGGAGAACTTCTAGTATATCTACATACCTAATATTATTGCCTTATTAAAAATGGAATCCCAACAATTACATCAAAATCCACATTCTCTTCAAAATCAATTGTCCTGTACTTCCTTGTTCATGTGTGTTCA AAAACGTTATATTTATAGGATAATTATACTCTATTTCTCAACAAGTAATTGGTTGTTTGGCCGAGCGGTCTAAGGCGCCTGATTCAAGAAATATCTTGACCGCAGTTAACTGTGGGAATACTCAGGTATC GTAAGATGCAAGAGTTCGAATCTCTTAGCAACCATTATTTTTTTCCTCAACATAACGAGAACACACAGGGGCGCTATCGCACAGAATCAAATTCGATGACTGGAAATTTTTTGTTAATTTCAGAGGTCGC CTGACGCATATACCTTTTTCAACTGAAAAATTGGGAGAAAAAGGAAAGGTGAGAGCGCCGGAACCGGCTTTTCATATAGAATAGAGAAGCGTTCATGACTAAATGCTTGCATCACAATACTTGAAGTTGA CAATATTATTTAAGGACCTATTGTTTTTTCCAATAGGTGGTTAGCAATCGTCTTACTTTCTAACTTTTCTTACCTTTTACATTTCAGCAATATATATATATATATTTCAAGGATATACCATTCTAATGTC TGCCCCTAAGAAGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAAGAAATCACAGCCGAAGCCATTAAGGTTCTTAAAGCTATTTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTA ATTGGTGGTGCTGCTATCGATGCTACAGGTGTTCCACTTCCAGATGAGGCGCTGGAAGCCTCCAAGAAGGCTGATGCCGTTTTGTTAGGTGCTGTGGGTGGTCCTAAATGGGGTACCGGTAGTGTTAGAC CTGAACAAGGTTTACTAAAAATCCGTAAAGAACTTCAATTGTACGCCAACTTAAGACCATGTAACTTTGCATCCGACTCTCTTTTAGACTTATCTCCAATCAAGCCACAATTTGCTAAAGGTACTGACTT CGTTGTTGTCAGAGAATTAGTGGGAGGTATTTACTTTGGTAAGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGATAGTGAACAATACACCGTTCCAGAAGTGCAAAGAATCACAAGAATGGCCGCT TTCATGGCCCTACAACATGAGCCACCATTGCCTATTTGGTCCTTGGATAAAGCTAATGTTTTGGCCTCTTCAAGATTATGGAGAAAAACTGTGGAGGAAACCATCAAGAACGAGTTCCCTACATTGAAGG TTCAACATCAATTGATTGATTCTGCCGCCATGATCCTAGTTAAGAACCCAACCCACCTAAATGGTATTATAATCACCAGCAACATGTTTGGTGACATCATCTCCGATGAAGCCTCCGTTATCCCAGGTTC CTTGGGTTTGTTGCCATCTGCGTCCTTGGCCTCTTTGCCAGACAAGAACACCGCATTTGGTTTGTACGAACCATGCCACGGTTCTGCTCCAGATTTGCCAAAGAATAAGGTCAACCCTATCGCCACTATC TTGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAGGCCATTGAAGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTAGGTGGTTCCAACAGTACCA CCGAAGTCGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCCTTGCTTAAAAAGATTCTCTTTTTTTATGATATTTGTACATAAACTTTATAAATGAAATTCATAATAGAAACGACACGAAATTACAAAA TGGAATATGTTCATAGGGTAGACGAAACTATATACGCAATCTACATACATTTATCAAGAAGGAGAAAAAGGAGGATGTAAAGGAATACAGGTAAGCAAATTGATACTAATGGCTCAACGTGATAAGGAAA AAGAATTGCACTTTAACATTAATATTGACAAGGAGGAGGGCACCACACAAAAAGTTAGGTGTAACAGAAAATCATGAAACTATGATTCCTAATTTATATATTGGAGGATTTTCTCTAAAAAAAAAAAAAT ACAACAAATAAAAAACACTCAATGACCTGACCATTTGATGGAGTTTAAGTCAATACCTTCTTGAACCATTTCCCATAATGGTGAAAGTTCCCTCAAGAATTTTACTCTGTCAGAAACGGCCTTAACGACG TAGTCGATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAG CTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGAC GTGCGGCCGCTCTAGAACTAGTGGATCAATTCCACGGACTATAGACTATACTAGTATACTCCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGTCCTTTAACGAGGCCTTACCACTCTTTTGTTAC TCTATTGATCCAGCTCAGCAAAGGCAGTGTGATCTAAGATTCTATCTTCGCGATGTAGTAAAACTAGCTAGACCGAGAAAGAGACTAGAAATGCAAAAGGCACTTCTACAATGGCTGCCATCATTATTAT CCGATGTGACGCTGCAGCTTCTCAATGATATTCGAATACGCTTTGAGGAGATACAGCCTAATATCCGACAAACTGTTTTACAGATTTACGATCGTACTTGTTACCCATCATTGAATTTTGAACATCCGAA CCTGGGAGTTTTCCCTGAAACAGATAGTATATTTGAACCTGTATAATAATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTATTTCGGTTCCTGGAGAAACTATTGCATCTATTGCATAGGTAATC TTGCACGTCGCATCCCCGGTTCATTTTCTGCGTTTCCATCTTGCACTTCAATAGCATATCTTTGTTAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAA CAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAG AATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTT AGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACT CCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATG ATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTG TCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACT TTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTAT ACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGC CTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGG GGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATGCATAGTACCGAGAAAC TAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGATGAGTATACGTTGTCCTGGCCACGGCAGAAGCACGCTTATCGCTCCAATTTCCCACAACATTAGTCAACTCCGTTAGGCCCTTCATTGAAAGA AATGAGGTCATCAAATGTCTTCCAATGTGAGATTTTGGGCCATTTTTTATAGCAAAGATTGAATAAGGCGCATTTTTCTTCAAAGCTGCGGCCGCACTCTCACTAGTACGTCAGGTGGCACTTTTCGGGG AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTC CGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCA ACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCAT ACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTT CTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCA ACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGA TCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAAT CTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTG TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGAT AGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGA AGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGT CGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTG TGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGT ATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGTACCGGGCCGGCCGTCGAGCTTGA TGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC GCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCA CATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTGGGGATCTACGTATGGTCATTTCTTCT TCAGATTCCCTCATGGAGAAAGTGCGGCAGATGTATATGACAGAGTCGCCAGTTTCCAAGAGACTTTATTCAGGCACTTCCATGATAGGCAAGAGAGAAGACCCAGAGATGTTGTTGTCCTAGTTACACA TGGTATTTATTCCAGAGTATTCCTGATGAAATGGTTTAGATGGACATACGAAGAGTTTGAATCGTTTACCAATGTTCCTAACGGGAGCGTAATGGTGATGGAACTGGACGAATCCATCAATAGATACGTC CTGAGGACCGTGCTACCCAAATGGACTGATTGTGAGGGAGACCTAACTACATAGTGTTTAAAGATTACGGATATTTAACTTACTTAGAATAATGCCATTTTTTTGAGTTATAATAATCCTACGTTAGTGT GAGCGGGATTTAAACTGTGAGGACCTTAATACATTCAGACACTTCTGCGGTATCACCCTACTTATTCCCTTCGAGATTATATCTAGGAACCCATCAGGTTGGTGGAAGATTACCCGTTCTAAGACTTTTC AGCTTCCTCTATTGATGTTACACCTGGACACCCCTTTTCTGGCATCCAGTTTTTAATCTTCAGTGGCATGTGAGATTCTCCGAAATTAATTAAAGCAATCACACAATTCTCTCGGATACCACCTCGGTTG AAACTGACAGGTGGTTTGTTACGCATGCTAATGCAAAGGAGCCTATATACCTTTGGCTCGGCTGCTGTAACAGGGAATATAAAGGGCAGCATAATTTAGGAGTTTAGTGAACTTGCAACATTTACTATTT TCCCTTCTTACGTAAATATTTTTCTTTTTAATTCTAAATCAATCTTTTTCAATTTTTTGTTTGTATTCTTTTCTTGCTTAAATCTATAACTACAAAAAACACATACAG 3

4 SUPPLEMENTAL FIGURES FIG S1 Plasmid map of p-luorin-lexa-ad and pmv118. 4

5 s F (45 nm) s A B C 1 3 Ratio F (45 nm) F (488 nm) F (488 nm) R(F45-nm/F488-nm) FIG S2 Ratiometric flow cytometry of luorin2. Yeast H439 cells were diluted with 2-ml spent medium and analyzed at low rate of 4 cells s -1. (A) The emission signals after 45- and 488-nm excitation were collected and plotted as pulse height signals. (B) A 2D plot of the fluorescence of F 45-nm versus F 488-nm and (C) a histogram of the fluorescence intensity ratios of F 45-nm /F 488-nm. 5

6 log ((Ri - R max )/(R min - Ri)) H H437 (15 min after nigericin addition) log R' = -.87(.4) + 6.2(.3) pka = 7. r 2 =.99 H437 (15 min after nigericin addition) log R' = -.9(.11) + 6.4(.7) pka = 7.1 r 2 =.95 H437 (1 h after nigericin addition) log R' = -.84(.6) + 6.(.4) pka = 7.1 r 2 =.98 H437 (2 h after nigericin addition) log R' = -.72(.5) + 5.2(.3) pka = 7.2 r 2 =.98 H437 (before nigericin addition) FIG S3 Reproducibility of calibrations for luorin2. Each calibration curve was calculated from the results of an individual set of experiments with different incubation periods and nigericin addition. A slope, an intercept, and pk a for each curve are depicted (right). Yeast H439 cells were washed, resuspended in calibration buffers, and treated with freshly prepared nigericin (1 µm). The cells were then analyzed at low rate of 4 cells s -1. For each point at least 2, cells were analysed. 6

7 s H437 s H399 A Sample Name Sample NameMean Name Mean,, StdDev Mean StdDev, StdDev,,, 1,1 1,1,64,64 1,1,64,78,78,6,78,6,6,633,633,58,633,58,58,553,553,51,553,51,51 Ex. 45 nm Ex. 476 nm,489,489,5,489,5,5 Pseudocolored,395,395,5,395,5,5,344,344,53,344,53,53,329,329,48,329,48, H B FL1:: FL1:: Sample - - FL1:: Name - F (45 nm) y5 nomtreated y5 y5 nomtreated 5.FCS 5.FCS 5.FCS Q2: Q2: -+, , Q2: -+, FL6:: FL6:: FL6:: 64-4 F (488 nm) Sample AlgParm Name AlgParm R(F45-nm/F488-nm) Y437 NT-23.FCS 25 Q2: -+, y5 nomtreated y5 y5 nomtreated 5.FCS 5.FCS 5.FCS y5 nomtreated y5 y5 nomtreated 5.FCS 5.FCS 5.FCS 2 Q2: Q2: -+, , Q2: -+, Q2: Q2: -+, , Q2: -+, Y437 NT-23.FCS Q2: -+, AlgParm AlgParm AlgParm 5 4 H Sample Name Y437 NT-23.FCS Q2: -+, FL1:: AlgParm FL6:: 64-4 F (45 nm) FL1:: - F (488 nm) AlgParm FL6:: 64-4 R(F45-nm/F488-nm) NT-2.FCS, Y437 NT-2.FCS Y437 NT-2.FCS Y437 NT-2.FCS 5 Q2: -+, Q2: -+, Q2: -+, FIG S4 The fluorescence ratio of luorin expressed from (A) an autonomously replicating plasmid (H399) is similar to that of (B) luorin2 with the gene integrated into a genome (H437). (A) Microscopic images at indicated excitation points (above) and peak width of florescence detected with a flow cytometer (below) show unequal expression of luorin among H399 cells. Despite this result, the pseudocolored images calculated from images taken at 45- and 476-nm excitation and the ratio of the fluorescence of F 45-nm /F 488-nm showed a high degree of i homogeneity (right panels), similar to that observed for H437 (right panel, B). 7

8 F (45 nm) s log ((Ri - R max )/(R min - Ri)) A 1 5 H F (488 nm) B R(F45-nm/F488-nm) C R (F45-nm / F488-nm) Sample Name Mean, StdDev, 1,1,64,78,6,633,58,553,51,489,5,395,5,344,53,329, H D 2 1 H H399 (15 min after nigericin addition) log R' = -1.7(.11) + (.7) pka = 7. r 2 =.96 H399 (1 h after nigericin addition) log R' = -.95(.7) + 6.8(.4) pka = 7.1 r 2 =.99 FIG S5 luorin behaved similarly to luorin2. (A) An overlay of the 2D plots of the fluorescence of F 45-nm versus F 488-nm and (B) the ratios of F 45-nm /F 488-nm showed correlation between and fluorescence. Yeast H399 cells were washed, resuspended in calibration buffers, and treated with nigericin for 15 min before analysis by flow cytometry. (C) Ratios at a given and (D) calibration curves of luorin are shown for cells treated with nigericin for 15 min (black) and 1 h (violet). The pk a 7.1 (±.1) of luorin calculated from calibration is similar to that of luorin2. 8

9 log ((Ri - R max )/(R min - Ri)) log ((Ri - R max )/(R min - Ri)) A B Ex. 45 nm Ex. 476 nm Pseudocolored C R (F45-nm/F476-nm) H D 3 2 H H E R (F45-nm / F476-nm) F H H399 H H399 log R' = -1.1(.5) + 7.4(.3) pkd = 7. (.1) r 2 =.99 H437 log R' = -.94(.5) + 6.6(.4) pkd = 7.1 (.1) r 2 =.98 FIG S6 In situ calibration curve of luorin2, determined by sequential scanning confocal microscopy. (A) Microscopic setup for imaging ratiometric probes, such as luorin, luorin2, or RaVC. (B) Pseudocolored images of H437-permeabilized cells calculated from images taken at 45- and 476-nm excitation. Different colors were assigned to defined values in accordance with the in situ calibration curve calculated with Eq. 2. (C) Plot of the fluorescence ratio versus. Yeast S. cerevisiae H437 cells expressing luorin2 were washed and resuspended in calibration buffers, and treated with freshly prepared nigericin to equalize ex with i. The ratio (R i ) between the emission intensities (collected at 5 to 55 nm) at 45- and 476-nm was calculated for each image using Eq. 1. (D) Logarithmic plot of the fluorescence intensities ratio (C) versus i. (E and F) In situ calibration curve of luorin. Yeast S. cerevisiae H399 cells expressing luorin were treated in a similar manner as H437, washed and resuspended in calibration buffers, and treated with freshly prepared nigericin. (E) Ratio of intensities and (F) logarithmic plot of ratio versus for H399 and H437 (presented in grey). 9

10 A B i8. pulse of glucose i 8. 5 mm sorbic acid 8. 1 mm sorbic acid FIG S7 Dynamic changes in i. (A) Yeast H437 grown for 1 h in SC-Met medium without glucose was fed with 2 g l -1 glucose; i was monitored by microscopy. i changes over 5 min after glucose pulsing are shown. (B) Yeast H437 cells grown in SC-Met with 2 g l -1 glucose were treated with 5 or 1 mm sorbic acid. i changes over 5 min were assessed by microscopy. i was calculated from images taken at 45- and 476-nm excitation at indicated time points. The arrow indicates glucose or sorbic acid addition. 1

11 A i 8. B C 2 g l -1 glycerol 2 g l -1 ethanol 2 g l -1 glucose FIG S8 The dynamics of the homeostasis of stationary cells was analyzed by confocal microscopy after feeding with 2 g l -1 (A) glycerol, (B) ethanol, or (C) glucose. The i and ratio (R i ) of the emission intensities (collected at 5 to 55 nm) at 45- and 476-nm excitation for each image were calculated. An average value (R i and i ) was calculated from at least 5 cells per image. After nutrient feeding, images at 45- and 476-nm excitation were taken every 2 s. The observed dynamics of homeostasis is similar to that determined by cytometry after glycerol, ethanol, or glucose feeding. The assessed i values present an average i for the populations, which differs from the cytometry-obtained i of the subpopulations. 11