PMID- 28518171
OWN - NLM
STAT- MEDLINE
DCOM- 20170710
LR  - 20181113
IS  - 1750-2799 (Electronic)
IS  - 1754-2189 (Print)
IS  - 1750-2799 (Linking)
VI  - 12
IP  - 6
DP  - 2017 Jun
TI  - FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single 
      cells using fluorescence in situ hybridization and flow cytometry.
PG  - 1245-1260
LID - 10.1038/nprot.2017.039 [doi]
AB  - We describe a flow-cytometry-based protocol for intracellular mRNA measurements 
      in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) 
      probes. The method, which we call FISH-Flow, allows for high-throughput 
      multiparametric measurements of gene expression, a task that was not feasible 
      with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell 
      fixation, permeabilization and hybridization with a set of fluorescently labeled 
      oligonucleotide probes. In this protocol, surface and intracellular protein 
      markers can also be stained with fluorescently labeled antibodies for 
      simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube 
      version of the protocol can be performed with a commercially available cell-wash 
      device that reduces cell loss, operator time and interoperator variability. It 
      takes  approximately 30 h to perform this protocol. An example of FISH-Flow measurements of 
      cytokine mRNA induction by ex vivo stimulation of primed T cells with specific 
      antigens is described.
FAU - Arrigucci, Riccardo
AU  - Arrigucci R
AD  - Public Health Research Institute, New Jersey Medical School, Rutgers, The State 
      University of New Jersey, Newark, New Jersey, USA.
FAU - Bushkin, Yuri
AU  - Bushkin Y
AD  - Public Health Research Institute, New Jersey Medical School, Rutgers, The State 
      University of New Jersey, Newark, New Jersey, USA.
FAU - Radford, Felix
AU  - Radford F
AD  - Public Health Research Institute, New Jersey Medical School, Rutgers, The State 
      University of New Jersey, Newark, New Jersey, USA.
FAU - Lakehal, Karim
AU  - Lakehal K
AD  - Public Health Research Institute, New Jersey Medical School, Rutgers, The State 
      University of New Jersey, Newark, New Jersey, USA.
FAU - Vir, Pooja
AU  - Vir P
AD  - Public Health Research Institute, New Jersey Medical School, Rutgers, The State 
      University of New Jersey, Newark, New Jersey, USA.
FAU - Pine, Richard
AU  - Pine R
AD  - Public Health Research Institute, New Jersey Medical School, Rutgers, The State 
      University of New Jersey, Newark, New Jersey, USA.
FAU - Martin, December
AU  - Martin D
AD  - BD Biosciences, San Jose, California, USA.
FAU - Sugarman, Jeffrey
AU  - Sugarman J
AD  - BD Biosciences, San Jose, California, USA.
FAU - Zhao, Yanlin
AU  - Zhao Y
AD  - Center for Immunity and Inflammation, New Jersey Medical School, Rutgers, The 
      State University of New Jersey, Newark, New Jersey, USA.
FAU - Yap, George S
AU  - Yap GS
AD  - Center for Immunity and Inflammation, New Jersey Medical School, Rutgers, The 
      State University of New Jersey, Newark, New Jersey, USA.
FAU - Lardizabal, Alfred A
AU  - Lardizabal AA
AD  - Global Tuberculosis Institute, New Jersey Medical School, Rutgers, The State 
      University of New Jersey, Newark, New Jersey, USA.
FAU - Tyagi, Sanjay
AU  - Tyagi S
AD  - Public Health Research Institute, New Jersey Medical School, Rutgers, The State 
      University of New Jersey, Newark, New Jersey, USA.
FAU - Gennaro, Maria Laura
AU  - Gennaro ML
AD  - Public Health Research Institute, New Jersey Medical School, Rutgers, The State 
      University of New Jersey, Newark, New Jersey, USA.
LA  - eng
GR  - R01 AI104615/AI/NIAID NIH HHS/United States
GR  - R01 AI106036/AI/NIAID NIH HHS/United States
GR  - R56 AI124691/AI/NIAID NIH HHS/United States
PT  - Journal Article
DEP - 20170518
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
RN  - 0 (Proteins)
RN  - 0 (RNA, Messenger)
SB  - IM
MH  - Automation, Laboratory/methods
MH  - Flow Cytometry/*methods
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Leukocytes, Mononuclear/chemistry
MH  - Proteins/*analysis
MH  - RNA, Messenger/*analysis
MH  - Single-Cell Analysis/*methods
PMC - PMC5548662
MID - NIHMS889170
EDAT- 2017/05/19 06:00
MHDA- 2017/07/14 06:00
PMCR- 2017/08/09
CRDT- 2017/05/19 06:00
PHST- 2017/05/19 06:00 [entrez]
PHST- 2017/05/19 06:00 [pubmed]
PHST- 2017/07/14 06:00 [medline]
PHST- 2017/08/09 00:00 [pmc-release]
AID - nprot.2017.039 [pii]
AID - 10.1038/nprot.2017.039 [doi]
PST - ppublish
SO  - Nat Protoc. 2017 Jun;12(6):1245-1260. doi: 10.1038/nprot.2017.039. Epub 2017 May 
      18.