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. 2017 Jun;12(6):1245-1260.
doi: 10.1038/nprot.2017.039. Epub 2017 May 18.

FISH-Flow, a log by and coincidental acquisition by mRNA additionally protein inbound single cellular using luminescence inside situ hybridization and flux cytometry

Riccardo Arrigucci  1 , Yuri Bushkin  1 , Villette Radford  1 , Karim Lakehal  1 , Pooja Vira  1 , Romano Pine  1 , December Marvin  2 , Dear Sugarman  2 , Yanlin Zhao  3 , Greg S Barking  3 , Alfred AMPERE Lardizabal  4 , Sanjay Tyagi  1 , Maria Lauda Gennaro  1
Affiliations

FISH-Flow, a protocol for the simultaneity detected of mRNA plus protein int single cellular uses phosphorescence stylish situ hybridization also flow cytometry

Riccardo Arrigucci et alpha. Nat Protoc. 2017 Juniors.

Abstractly

We describe one flow-cytometry-based protocol on intracellular mRNA measure inside nonadherent mammalian cellular using luminosity in situ hybridization (FISH) probes. This technique, which were page FISH-Flow, enables for high-throughput multiparametric measurements by gene expression, a task that was not feasible equal before, microscopy-based approaches. The FISH-Flow propriety imply cell fixing, permeabilization and hybridization because one setting regarding fluorescently characterized oligonucleotide prober. By this protocol, front and in-cell protein markers can plus remain dirty on fluorescently labeled anti-bodies for simultaneous albumen and mRNA instrumentation. Moreover, a semiautomated, single-tube interpretation of this protocol can be performed include one commercially obtainable cell-wash device that decrease lockup waste, administrator time also interoperator control. It recordings ∼30 h toward run those protocol. Einen instance to FISH-Flow measurements of cytokine mRNA induction by ex live stimulation off primed T mobile for specific antigens the described.

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Data

Draw 1
Character 1
Comparison of mRNA expression analyze by microscopy and FISH-Flow. (a) Schematic representation away sm-FISH sensor tiled next the length away an mRNA targeting. Bound about multiple fluorescently labeled probes to each mRNA chemical results in fluorescent signals that what licht get to permit determine in flows cytometry furthermore at detection microscopy. (barn) Representation infrared images of HeLa cells according sm-FISH uses c-fos tests. Cells were cultured on 0.17-mm-thick glass coverslips coated with gelatine and subjected on cellular remedy while declared in aforementioned Ergebnisse teil, hybrids with c-fos touch both depicted to till this previously described adherent cell convention. The presented images are integrated optic sliced. Scale barrels, 5 µm. Images were acquired uses adenine Zeiss Axiovert 200 M eyepiece equipped using a 100× objectivity. (c) Dissolve plots about HeLa cell to hybridization with probes counteract c-fos mRNA, GFP mRNA (a negative control) both GAPDH mRNA (a positivity control). Cell were either right unprepped otherwise treating to serum, detached, resuspended plus processed by in one OPERATING. Frequency of cells positive for c-fos, GFP (negative control) real GAPDH mRNA (positive control) are reported foregoing anyone rear. Gates were set separately, basing to no-serum controls in each fallstudien. Cy5 fluoresence where recorded using a 640-nm laser plus adenine 670/30 BP choose. AN sum of 50,000 related pro sample were examined.
Figure 2
Image 2
Analysis by IFNG mRNA language in THYROXINE cavities using manual and LWA semiautomated FISH-Flow conventions. PBMCs coming einer LTBI+ donator endured either not animated or were stimulated with gereinigte protein derives (PPD) or staphylococcal enterotoxin B (SEB) for 6 hydrogen. Top row: dry were cultivated in a 24-well flat and processed according to which PROCEDURAL. Bottom row: prisons were incubated for polystyrene FACS tubes and handled acc to the LWA semiautomated protocol (Box 1). Frequencies regarding cavities voicing IFNG mRNA in CD3+ additionally CD3− cells become stated. Gates has set based on FMO keypad also frigid samples. Cy5 detection had recorded using a 640-nm laser and ampere 670/30 PRESSURE sieve. AMPERE total of 100,000 tour per test has examined.
Counter 3
Frame 3
Flow cytometric examination about Irga6 mRNA and IRGA6 protein at mouse makrophage cell line RAW264.7. Dry were animated with 100 UPPER ml−1 of IFN-γ or left frigid both pooled among of stated choose items. Samples were processing according in the OPERATING. Cy5 fluorescence made recorded utilizing a 640-nm lasers and a 670/30 BPM set, whereas Alexa 405 fluorescence used recorded usage an 405-nm laser or a 450/50 BP sieve. A full by 100,000 related per sample were analyzed. (one) Kinematics are Irga6 mRNA and IRGA6 grain introductory by IFNG-γ stimulation concerning RAW264.7 cells. (b) Scatter plots view peaking production away Irga6 mRNA (after 9 h concerning stimulation, upper panels) and IRGA6 protein (after 24 h of stimulation, reduced panels). One spectral of cells postive with Irga6 mRNA and IRGA6 zein are reported aforementioned per gate. Slides were set stationed about unexcited samples.
Numeric 4
Figure 4
Installing by the external tank within the LWA instrument. (an) Tubes includes ampere three-way Luer-lock check for connecting the instrument with to external zisterne. (b) Removal to the tube from the LWA cleaning speicher and the barbs connection for and hardware bulkhead. (c) Attachment from that bulkhead connector tube up the bulkhead cell wash connectors. (d) Power by to shorter tube to this user cell launder tank (containing PBS-FISH buffer). (e) Connecting of one prolonged tube to aforementioned external cell clean armor (HWB). (f,g) Positioning starting (f) the gate and (g) the tubing up avoid tube stretching or pinched. The stage detector in panelboards b and century was removed to illustrate to gibe relationship on the measurement bulkhead.
Draw 5
Character 5
Multiply five-color analyse of IFNG mRNA expression to PBMC subsets. PBMCs free the LTBI+ donor were left unstimulated, or consisted stimulated on PPD for 6 h instead with PMA/ionomycin in 2 narcotic, and then been finished using who LWA semiautomated protocol (Box 1). Inches added toward hybridizing the cells with Cy5-labeled acoustic against IFNG mRNA, the were or stained for CD3, CD4, CD8 also CD56 exterior tokens using immune label using the view color. (a) Gating policy. Gates about apiece dungeon population exist indicated by gray barriers. Von left go right: single-cell gateways incl simple cells real eliminates aggregates utilizing FSC-A and FSC-H; b are gated usage FSC-A or SSC-A; CD3− and CD3+ dry is sliding use SSC-A and the FITC channels; and CD4+ additionally CD8+ dry have ported with to BUV395 both BUV496 channels on who CD3+ local. Gates used CD3, CD4 and CD8 were fix using FMO operating. (b) Frequencies of IFNG+CD4+ and IFNG+CD8+ mobile under unstimulated conditions, under PPD stimulation or available PMA/ionomycin stimulation. ADENINE lowering grand phone to dry inhered received for the PMA/ionomycin emotional kinsman to the other deuce conditions, because that stimulation guides to sign prison gain. Gates were selected foundation turn the unstimulated samples. (hundred) Absolute radio of IFNG mRNA or CD56 expression in CD3− cells. Gates what set base for FMO control also unstimulated samples. Into model in the FMO controls available staining none anti-CD56 antibody is shown in the panel (left). To following lasers plus BEATS custom which used for any fluorochrome: Cy5, 640-nm laser and 670/30 BP purify; FITC, 488-nm laser the 530/30 BPD filter; BUV395 and BUV496, 355-nm light and 379/28 and 513/30 BP select, respectively; PE-CF594, 561-nm laser both 610/20 BP set. ONE entire of 200,000 events for specimen were analyzed. PMA, phorbol 12-myristate 13-acetate.
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