Phospho flowcytometri med fluorescerende celle Barcoding for enkelt celle signalisering analyse og biomarkør oppdagelsen
Summary
Her vises en protokoll for middels til høy gjennomstrømming analyse av protein fosforylering hendelser på cellenivå. Phospho-flowcytometri er en effektiv tilnærming til å karakterisere signalnettverk avvik, identifisere og validere biomarkers og vurdere pharmacodynamics.
Abstract
Avvikende celle signalisering spiller en sentral rolle i hudkreft og progresjon. Mest romanen målrettet terapi er faktisk rettet mot proteiner og protein funksjoner, og cellen signalnettverk avvik kan fungere som biomarkers å angi personlige behandlingstilbud. I motsetning til DNA og RNA analyser, kan endringer i protein aktivitet mer effektivt vurdere mekanismene bak narkotika følsomhet og motstand. Phospho-flowcytometri er en kraftfull teknikk som måler protein fosforylering hendelser på cellenivå, en viktig funksjon som skiller denne metoden fra andre antistoff tilnærminger. Metoden tillater samtidig analyse av flere signalnettverk proteiner. I kombinasjon med fluorescerende celle barcoding, kan større middels til høy gjennomstrømming datasett erverves av standard cytometer på kort tid. Phospho flowcytometri har programmer både studier av grunnleggende biologi og klinisk forskning, inkludert signalering analyse, biomarkør oppdagelse og vurdering av pharmacodynamics. Her tilbys en detaljert eksperimentelle protokoll for phospho flyt analyse av renset perifert blod mononukleære celler, med kronisk lymfatisk leukemi celler som eksempel.
Introduction
Phospho-flowcytometri brukes til å analysere protein fosforylering nivå encellede oppløsning. Det overordnede målet med metoden er å tilordne mobilnettet signalnettverk mønstre angitte vilkår. Ved å utnytte flowcytometri multiparameter kapasitet, kan flere signalveier analyseres samtidig i forskjellige undergrupper av en heterogen celle befolkningen som perifert blod. Disse trekkene tilbyr fordeler over andre antistoff-baserte teknologier som immunohistochemistry, enzym knyttet immunosorbent analysen (ELISA), protein matrise og omvendt fase protein matrise (RPPA)1. Phospho flowcytometri kan kombineres med fluorescerende celle barcoding (FCB), som betyr at enkeltcelle prøver er merket med unike signaturer av fluorescerende fargestoffer slik at de kan være blandet sammen, beiset og analyseres som en enkelt prøve2. Dette reduserer antistoff forbruket øker data robusthet gjennom kombinasjonen av kontroll og behandlet prøver og forbedrer hastigheten på oppkjøpet. Kombinert FCB befolkningen kan deretter delt inn i mindre prøver og farget med opp til 35 forskjellige phospho-spesifikke antistoffer, avhengig av starter materiale. Store profilering eksperimenter kan dermed kjøres med standard cytometer maskinvare. Phospho-flowcytometri brukes på profil signalnettverk trasé i pasientprøvene fra flere Hematologisk kreft inkludert kronisk lymfatisk leukemi (CLL)3,4,5, akutt myelogen leukemi (AML) 6 og non-Hodgkin lymfomer7. Phospho flowcytometri er dermed en effektiv tilnærming til å karakterisere signalnettverk avvik, identifisere og validere biomarkers og vurdere pharmacodynamics.
Her, tilbys optimalisert protokollen for analyse av CLL pasientprøvene av phospho flowcytometri (figur 1A). Eksempler på basale signalnettverk karakterisering, anti-IgM/B celle reseptor stimulering og narkotika forstyrrelsene vises. En detaljert beskrivelse av en FCB matrise tilbys. Protokollen kan enkelt tilpasses til andre suspensjon celletyper.
Protocol
Representative Results
Discussion
Phospho-flowcytometri er en kraftfull teknikk å måle protein fosforylering nivå i enkeltceller. Siden metoden er avhengig av farging med antistoffer, begrenses phospho flowcytometri antistoff-tilgjengelighet. Videre for å få pålitelige resultater, bør alle antistoffer titreres og kontrollert før bruk. En detaljert protokoll for titrering av phospho-spesifikke antistoffer er beskrevet andre steder12. Under panelet design er vurdering av signal-til-støy forholdet avgjørende. I eksemplet pr…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbeidet ble utført i laboratoriet av Professor Kjetil Taskén, og ble støttet av den norske Kreftforening og Stiftelsen Kristian Gerhard Jebsen. Johannes Landskron og Marianne Enger er anerkjent for kritisk lesing av manuskriptet.
Materials
| RPMI 1640 GlutaMAX | ThermoFisher Scientific | 61870-010 | Cell culture medium |
| Fetal bovine serum | ThermoFisher Scientific | 10270169 | Additive to cell culture medium |
| Sodium pyruvate | ThermoFisher Scientific | 11360-039 | Additive to cell culture medium |
| MEM non-essential amino acids | ThermoFisher Scientific | 11140-035 | Additive to cell culture medium |
| Lymphoprep | Alere Technologies AS | 1114547 | Density gradient medium |
| Anti-IgM | Southern Biotech | 2022-01 | For stimulation of the B cell receptor |
| BD Phosflow Fix Buffer I | BD | 557870 | Fixation buffer |
| BD Phosflow Perm Buffer III | BD | 558050 | Permeabilization buffer |
| Alexa Fluor 488 5-TFP | ThermoFisher Scientific | A30005 | Barcoding reagent |
| Pacific Blue Succinimidyl Ester | ThermoFisher Scientific | P10163 | Barcoding reagent |
| Pacific Orange Succinimidyl Ester, Triethylammonium Salt | ThermoFisher Scientific | P30253 | Barcoding reagent |
| Compensation beads | Defined by user | Correct species reactivity | |
| Falcon tubes | Defined by user | ||
| Eppendorf tubes | Defined by user | ||
| 96 well V-bottom plates | Defined by user | Compatible with the flow cytometer | |
| Centrifuges | Defined by user | For Eppendorf tubes, Falcon tubes and plates | |
| Water bath | Defined by user | Temperature regulated | |
| Flow cytometer | Defined by user | With High Throughput Sampler (HTS) | |
| Name | Company | Catalog Number | Comments |
| Antigen | |||
| AKT (pS473) | Cell Signaling Technologies | 4075 | Clone: D9E Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 |
| ATF-2 (pT71) | Santa Cruz Biotechnology | sc-8398 | Clone: F-1 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
| BLNK (pY84) | Beckton Dickinson Pharmingen | 558443 | Clone: J117-1278 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
| Btk (pY223)/Itk (pY180) | Beckton Dickinson Pharmingen | 564846 | Clone: N35-86 Reference: Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
| Btk (pY551) | Beckton Dickinson Pharmingen | 558129 | Clone: 24a/BTK (Y551) Reference: Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 |
| Btk (pY551)/Itk (pY511) | Beckton Dickinson Pharmingen | 558134 | Clone: 24a/BTK (Y551) Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 |
| CD3ζ (pY142) | Beckton Dickinson Pharmingen | 558489 | Clone: K25-407.69 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 |
| Histone H3 (pS10) | Cell Signaling Technologies | 9716 | Clone: D2C8 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
| IκBα | Cell Signaling Technologies | 5743 | Clone: L35A5 Reference: Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
| LAT (pY171) | Beckton Dickinson Pharmingen | 558518 | Clone: I58-1169 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 |
| Lck (pY505) | Beckton Dickinson Pharmingen | 558577 | Clone: 4/LCK-Y505 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
| MEK1 (pS298) | Beckton Dickinson Pharmingen | 560043 | Clone: J114-64 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 |
| NF-κB p65 (pS529) | Beckton Dickinson Pharmingen | 558422 | Clone: K10-895.12.50 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
| NF-κB p65 (pS536) | Cell Signaling Technologies | 4887 | Clone: 93H1 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 |
| p38 MAPK (pT180/Y182) | Cell Signaling Technologies | 4552 | Clone: 28B10 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
| p44/42 MAPK (pT202/Y204) | Cell Signaling Technologies | 4375 | Clone: E10 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
| p53 (pS15) | Cell Signaling Technologies | NN | Clone: 16G8 Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
| p53 (pS20) | Cell Signaling Technologies | NN | Clone: Polyclonal Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
| p53 (pS37) | Cell Signaling Technologies | NN | Clone: Polyclonal Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
| p53 (pS46) | Cell Signaling Technologies | NN | Clone: Polyclonal Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
| p53 (pS392) | Cell Signaling Technologies | NN | Clone: Polyclonal Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
| PLCγ2 (pY759) | Beckton Dickinson Pharmingen | 558498 | Clone: K86-689.37 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
| Rb (pS807/pS811) | Beckton Dickinson Pharmingen | 558590 | Clone: J112-906 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
| S6-Ribos. Prot. (pS235/236) | Cell Signaling Technologies | 4851 | Clone: D57.2.2E Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
| SAPK/JNK (pT183/Y185) | Cell Signaling Technologies | 9257 | Clone: G9 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
| SLP76 (pY128) | Beckton Dickinson Pharmingen | 558438 | Clone: J141-668.36.58 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 |
| STAT1 (pY701) | Beckton Dickinson Pharmingen | 612597 | Clone: 4a Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
| STAT3 (pY705) | Beckton Dickinson Pharmingen | 557815 | Clone: 4/P-STAT3 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
| STAT4 (pY693) | Zymed/ThermoFisher Scientific | 71-7900 | Clone: Polyclonal Reference: Uzel et al., 2001, Detection of intracellular phosphorylated STAT-4 by flow cytometry, Clin Immunol, 100(3): 270-6 |
| STAT5 (pY694) | Beckton Dickinson Pharmingen | 612599 | Clone: 47/Stat5(pY694) Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
| STAT6 (pY641) | Beckton Dickinson Pharmingen | 612601 | Clone: 18/P-Stat6 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
| SYK (pY525/Y526) | Cell Signaling Technologies | 12081 | Clone: C87C1 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 |
| ZAP70/SYK (pY319/Y352) | Beckton Dickinson Pharmingen | 557817 | Clone: 17A/P-ZAP70 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
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