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Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.
The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.
Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) are central regulators of necroptotic cell death1,2. Necroptosis is a lytic and inflammatory form of regulated cell death involved in antiviral immunity and autoinflammation. Necroptosis of virus-infected cells immediately shuts down virus replication. Cell lysis following necroptosis induction also releases damage-associated molecular patterns, which stimulate antiviral immunity3,4. Necroptosis is initiated by the activation of RIPK3 following RI....
1. Preparation of biotinylated tyramide
2. Maintaining HT-29 cells in culture
NOTE: ZBP1-expressing HT-29 were generated by transduction with a lentivector27 encodi.......
The immunofluorescent detection of MLKL phosphorylation and especially RIPK3 phosphorylation in human cells is technically challenging26. We here present an improved staining protocol for human p-RIPK3 (S227) and p-MLKL (S358) upon the activation of ZBP1. The protocol includes a TSA step to improve the detection limit and sensitivity of the fluorescent signals. To validate the method, a side-by-side comparison of the TSA-mediated immunofluorescence with standard indirect fluorescent staining of bo.......
This immunofluorescent staining protocol describes the use of tyramide signal amplification (TSA) to increase the sensitivity for signaling events of the human necroptotic signaling pathway that are difficult to detect, including the phosphorylation of RIPK3 and MLKL26. The inclusion of a TSA step significantly improves the detection threshold of p-RIPK3 (S227) and p-MLKL (S358) and increases the sensitivity of p-MLKL (S358) straining. TSA revealed a p-RIPK3 (S227) signal already present in the mo.......
We would like to thank the VIB Bioimaging Core for training, support, and access to the instrument park. J.N. is supported by a PhD fellowship from the Research Foundation Flanders (FWO). Research in the J.M. group was supported by an Odysseus II Grant (G0H8618N), EOS INFLADIS (40007512), a junior research grant (G031022N) from the Research Foundation Flanders (FWO), a CRIG young investigator proof-of-concept grant, and by Ghent University. Research in the P.V. group was supported by EOS MODEL-IDI (30826052), EOS INFLADIS (40007512), FWO senior research grants (G.0C76.18N, G.0B71.18N, G.0B96.20N, G.0A9322N), Methusalem (BOF16/MET_V/007), iBOF20/IBF/039 ATLANTIS, ....
Name | Company | Catalog Number | Comments |
Antibodies | |||
Anti-rabbit HRP | Agilent Technologies Belgium | K4002 | Envision+ System-HRP Labelled Polymer anti-rabbit |
Goat anti-mouse DyLight 633 | Thermofisher | 35513 | Secundary antibody |
HSV-1 ICP0 | Santa Cruz | sc-53070 | Mouse anti-ICP0(HSV-1) antibody |
IAV-PR8 mouse serum | In house production | xx | Mouse anti-IAV-PR8 polyclonal antibody |
pMLKL | Abcam | ab187091 | Rabbit anti-MLKL-phospho S358 antibody |
pRIPK3 | Abcam | ab209384 | Rabbit anti-RIPK3-phospho S227 antibody |
Fluorophores | |||
DAPI | Thermofisher | D21490 | To visualise the nucleus of the cells |
Streptavidin coupled to Alexa Fluor 568 | Thermofisher | S11226 | To visulalise biotin molecules |
Compounds | |||
30% H2O2 | Sigma | H1009 | Oxidising substrate, necessary for HRP activity |
4% PFA | SANBIO | AR1068 | To fix/crosslink the cells |
Biotinyl-tyramide | R&D Systems | 6241 | To amplify signal, HRP substrate |
BV-6 | Selleckchem | S7597 | BV6 IAP Inhibitor |
  |   |   | For cell culture: to detach the cells |
  |   |   | 8.0 g/L NaCl |
  |   |   | 0.4 g/L disodium salt of EDTA |
EDTA 0.04% | In house formulation | 1.1 g/L Na2HPO4 | |
  |   |   | 0.2 g/L NaH2PO4 |
  |   |   | 0.2 g/L KCl |
  |   |   | 0.2 g/L Glucose |
Fetal Bovine serum | TICO | FBS EU XXX | For cell culture, maintaining cell culture; lot number: 90439 |
GSK'840 | Aobious | AOB0917 | RIPK3 kinase inhibitor |
L-Glutamine | Sigma-Aldrich | G7513 | For cell culture, maintaining cell culture |
MAXblock | Active Motif | 15252 | Blocking solution |
PBS | Gibco | 10444402 | |
Sodium pyruvate | Sigma-Aldrich | S8636 | For cell culture, maintaining cell culture |
TNF-α | In house production | - | Signaling molecule, able to trigger cell death in combination with BV6 and zVAD |
Triton X-100 | Sigma Aldrich | T8787-50ML | To permeabilise the cells |
Trypan blue | Merck | 11732 | For cell counting, used as live/dead marker at 0,1% |
Trypsine | Sigma-Aldrich | T4424 | For cell culture: to detach the cells |
zVAD | Bachem | BACE4026865.0005 | Z-Val-Ala-DL-Asp-fluoromethylketone |
Material | |||
µ-Slide 8 well high glass bottom | iBidi | 80807 | To culture the cells |
Cotton Preping Balls-size medium | Electron Microscopy Sciences | 71001-10 | To clean the objectives |
Immersol 518 F / 30 °C | ZEISS | 444970-9000-000 | To visualise the sample at high magnifications |
Lens Cleaner | ZEISS | 000000-0105-200 | To clean the objectives |
LSM880 Fast Airyscan confocal microscope | To visualise the sample | ||
Software | |||
Excel | Office | xx | To process the data |
Prism 9 | Graphpad | xx | To analyse the data- statistical testing and graph generation |
Volocity 6.3 | Volocity | xx | To perform quantifications |
Zen black | ZEISS | xx | To aquire and process images |
Zen blue | ZEISS | xx | To visualise images |
Viruses | |||
HSV-1 (mutRHIM) F strain | produced by Dr. Jiahuai Han | in house replication | HSV-1 as a trigger for necroptosis; RHIM core domain of UL39/ICP6 is mutated (VQCG>AAAA) |
HSV-1 (WT) F strain | Produced by Dr. Jiahuai Han | in house replication | HSV-1 (WT) as a negative control for necroptosis induction (ICP6 inhibition) |
IAV PR8 | in house stock | in house replication | IAV as a trigger for necroptosis |
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