The authors would like to acknowledge the Montpellier Rio Imaging (MRI) platform where the acquisitions were performed. They thank Isabel Cristina Lopez Mejia, Alexandra Metz, Irina Lassot, Solange Desagher, Fabien Loustalot, and Virginie Georget for their help in different parts of the protocol. This work was supported by the Fondation pour la Recherche M&#233;dicale (FRM) (Equipe FRM 2011 -n&#176;DEQ20111223745).

All the animal procedures in this protocol are in strict adherence with the guidelines of the European Community Council Directive of 24 November 1986 (86-609/EEC). House the mice in group, allowing food and water ad libitum. Maintain them in a controlled environment (22 &#177;1 &#176;C, 55 &#177;5% humidity) with a 12 hr:12 hr light:dark cycle (light on at 7:00 am).
1. Culture of Murine Primary Cells: Mouse Embryonic Fibroblasts (MEFs)
<ol>
	<li>Autoclave thin forceps, as well as curved forceps and dissecting scissors. Store in a sterile container. Use sterile D-PBS (Dulbecco&#39;s Phosphate-Buffered Saline). Prepare complete MEFs medium: Dulbecco&#39;s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal calf serum (FBS), 1 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, and 0.5 mM 2-mercaptoethanol and warm at 37 &#176;C.</li>
	<li>Euthanize a pregnant mouse (at 13.5 days post-co&#239;tum (dpc)) by cervical dislocation. Remove the uterine horns from the mouse sanitized with&#160;90% EtOH. Under a sterile and clean hood, place the horns with the embryos in 37 &#176;C prewarmed D-PBS. Open the horns, place the embryos in a 100 mm sterile plastic Petri dish, clean them up from the umbilical cord material and wash them with warm D-PBS to remove blood excess. Under a binocular, remove the embryo&#8217;s limbs, the internal organs and the upper part of the head containing the brain.</li>
	<li>In a new Petri dish, mince the embryos into very small pieces with a sterile surgical blade or scissors (at least for 1 min). In the case of embryos of different genotypes, be careful to put each embryo in individual Petri dishes and keep the tail or the upper head for genotyping.</li>
	<li>Cover the embryo with 1x&#160;trypsin-EDTA (0.25% trypsin, 1 mM EDTA) . Incubate for 30 min to 1 hr in a 37 &#176;C incubator. Add 10 ml of complete MEFs medium to stop trypsin reaction. Pipet up and down to remove all aggregates. Let sit the cell suspension into a 50 ml tube (filled with complete medium) for 10 min, and centrifuge the supernatant for 5 min at 300 x g at room temperature. Resuspend the pellet in complete medium (6 ml for 1 embryo). Viable nucleated cells can be counted using trypan blue (around 5 x 106 cells can be expected from one embryo). Plate 3 ml per 60 mm Petri dish.</li>
	<li>Change the medium the next day and allow the cells to grow until the dishes are confluent. Many cell types can be seen, but only fibroblasts will survive subculture. </li>
	<li>Split the cells and allow them to grow in 35 mm dishes with glass bottom (important for time lapse experiment), until 50-70% confluency for the transfection. Test for Mycoplasma and mouse pathogens. </li>
</ol>
2. Culture Adaptations in the Case of Neurons
<ol>
	<li>The day before the culture, coat 35 mm glass bottom Petri dishes with poly-L-lysine (200 &#181;l of 0.1 mg/ml) under a sterile and clean hood, and leave overnight.</li>
	<li>On the next morning, rinse with sterile pure water, twice for 5 min and once for 45 min to 1 hr. Replace with 2 ml of DMEM plus 10% FBS medium and keep in a 37 &#176;C incubator.</li>
	<li>Dissect embryos at 18.5 dpc. Under a sterile and clean hood, place the horns with the embryos in cold sterile HBSS (Hank&#39;s Balanced Salt Solution) in 100 mm Petri dish. Neonatal pups can also be used instead of embryos in order to preserve the life of the dam and enable it to produce more offspring, especially in the case of transgenic animals which can be difficult to obtain. In individual Petri dishes, take each embryo or newborn and cut the head with scissors. Hold the head by inserting curved forceps into the eyes, cut the skin and carefully open the soft skull from the back of the head until the eyes, on each side of the head. Cut the optic nerves and the brain stem, remove the brain, and put it in a new Petri dish containing HBSS. Under a stereoscope, remove all the meninges using two thin forceps. Separate the hippocampi, the cortex or any other part of the brain depending on the structure to study.</li>
	<li>Immerse the dissected brain structure in 4.5 ml of cold HBSS prepared previously in 15 ml tubes and keep on ice until digestion with trypsin. Add 0.5 ml of 2.5% trypsin and incubate at 37 &#176;C for 15 min to 20 min. Rinse the trypsin 3x&#160;with HBSS, being extremely careful to not discard the digested brain parts.</li>
	<li>Resuspend in 500 &#181;l (hippocampi) to 1 ml (cortex) DMEM plus 10% FBS and pipet up and down several times with a 1 ml micropipette equipped with a 1 ml tip, then equipped with 1 ml plus 200 &#181;l tips, until there is no visible aggregate.</li>
	<li>Distribute 100-200 &#181;l of cell suspension to each 35 mm glass bottom dish containing DMEM plus 10% FBS and let the neurons adhere at 37 &#176;C for at least 3 hr. Replace by prewarmed neuron complete medium (Neurobasal medium supplemented with 250 &#181;M L-glutamine and NS21, prepared as described in Chen et al.19) and leave at 37 &#176;C to allow neuronal growth. Transfect the neurons at 5 to 14 days in vitro (div) (the efficiency of transfection is higher after a couple of div but synaptic connections are better established from 7-10 div).</li>
</ol>
3. Transfection of EGFP-G3BP1 Construct
Transfect the cells with a vector containing the cDNA of your protein of interest (any component of SGs) fused to a fluorescent marker (GFP, YFP, etc.), using 3 &#181;g of purified plasmid per 35 mm dish.
<ol>
	<li>Transfect the MEFs using a commercial method, following the manufacturer&#39;s protocol (See Table of Materials/Reagents).</li>
	<li>Transfect the neurons with a calcium phosphate method adapted from Xia et al.20&#160;Briefly:
	<ol>
		<li>Prepare the solutions: DMEM-wash: DMEM containing 25 mM KCl; transfection solution: DMEM-wash containing 1x&#160;DMKY (HEPES 5 mM, MgCl2 10 mM, phenol red); and shock solution: HeBS 1x, DMKY 1x,&#160;and DMSO 2% (v/v); and keep them at 37 &#176;C.</li>
		<li>Remove the media from the neurons, filtrate, and keep it at 37 &#176;C. Wash with DMEM-wash then replace with transfection medium and keep at 37 &#176;C during the preparation of the calcium phosphate-plasmid DNA precipitates.</li>
		<li>In a 1.5 ml microcentrifuge tube, add (in this order) Braun water (final volume 50 &#181;l), 5 &#181;l of CaCl2 2.5 M, and 3 &#181;g of plasmid DNA. Drop this mix onto 50 &#181;l of HeBS 2x&#160;already introduced in a round bottom polypropylene tube. Mix the tube by rotation along with the dropping. Let the precipitate form at room temperature during 30 min.</li>
		<li>Add the precipitate dropwise onto the neurons and leave at 37 &#176;C during 30 to 50 min.</li>
		<li>Replace with shock solution for 1 min, then rinse with DMEM-wash and finally reintroduce the preconditioned medium. Keep the cells at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Visualization of G3BP Containing SGs Assembly and Dynamics
<ol>
	<li>Replace the medium of the cells which contains phenol red by phenol red-free medium.</li>
	<li>Use a confocal microscope equipped with fluorescence for the acquisitions. Turn on the microscope systems: mercury lamp, computer, and lasers. Warm up the chamber to 37 &#176;C at least 20 min before the beginning of the acquisitions.</li>
	<li>The over expression of G3BP induces the spontaneous formation of a type of SGs. The cells may thus be observed for the assembly of SGs 24 hr right after the transfection without stress induction. Alternatively, stress can be induced to further induce the assembly of SGs. Add 0.5 mM sodium arsenite and start the acquisition right after the addition of the compound (granules will be well formed within 1 hr).</li>
	<li>Add oil to the immersion objective (use 40X or 63X objectives) and install the Petri dish over the objective in the adapted 35 mm microscope holder, stabilized on the stage holder. Check that the dish is flat otherwise the acquisitions will be altered. Turn on the fluorescence light according to the relevant fluorophore and visualize transfected cells. Turn off fluorescence once a cell of interest is in the field in order to minimize bleaching and cytotoxicity.</li>
	<li>In the &#34;acquire tab&#34;, select the lasers depending on the excitation wavelength of the tag fluorophore (488 nm in our protocol which uses EGFP-tagged G3BP) and select the adapted emission length for the PMT (photomultiplier tube). Adjust the laser power (usually not more than 10%) to minimize noise and oversaturation as well as toxicity, and set the gain and offset to modify the signal to noise ratio. Scan fast (1,000 Hz) in order to minimize the duration of laser exposition (line average 2, frame average 1). If desired, set the parameters for the Z stack to be able to reconstruct the image in 3D (a Z step of 1 &#181;m is usually fine with cells). Determine the interval time between each acquisition: smaller intervals permit to obtain a more coherent movie but this may induce photobleaching and toxicity (an interval of 20 sec was used in the described experiments). Duration of total acquisition may vary depending on the stress type. Many G3BP-containing SGs are formed very rapidly under arsenite treatment and are well visible after 1 hr of treatment: in that case, choose rapidly the cells to film and start the acquisitions right after the stress, with a total duration of acquisitions of 15-20 min largely sufficient to observe the assembly of several SGs.</li>
	<li>Save the pictures and reconstruct the stacks and movie using ImageJ software.</li>
</ol>

<ol>
	<li>Thomas, M. G., Loschi, M., Desbats, M. A., Boccaccio, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+granules:+The+good,+the+bad+and+the+ugly.">RNA granules: The good, the bad and the ugly.</a> Cellular Signalling. 23 (2), 324-334 (2011).</li><li>Kedersha, N., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granules:+sites+of+mRNA+triage+that+regulate+mRNA+stability+and+translatability.">Stress granules: sites of mRNA triage that regulate mRNA stability and translatability.</a> Biochemical Society transactions. 30 (6), 963-969 (2002).</li><li>Anderson, P., Kedersha, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granules:+the+Tao+of+RNA+triage.">Stress granules: the Tao of RNA triage.</a> Trends in Biochemical Sciences. 33 (3), 141-150 (2008).</li><li>Gilks, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granule+assembly+is+mediated+by+prion-like+aggregation+of+TIA-1.">Stress granule assembly is mediated by prion-like aggregation of TIA-1.</a> Molecular biology of the cell. 15 (12), 5383-5398 (2004).</li><li>Tourri&#232;re, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RasGAP-associated+endoribonuclease+G3BP+assembles+stress+granules.">The RasGAP-associated endoribonuclease G3BP assembles stress granules.</a> The Journal of Cell Biology. 160 (6), 823-831 (2003).</li><li>Parker, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Ras-GTPase-activating+protein+SH3-domain-binding+protein.">A Ras-GTPase-activating protein SH3-domain-binding protein.</a> Molecular and Cellular Biology. 16 (6), 2561-2569 (1996).</li><li>Annibaldi, A., Dousse, A., Martin, S., Tazi, J., Widmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+G3BP1+as+a+RasGAP+binding+protein:+sensitization+of+tumor+cells+to+chemotherapy+by+the+RasGAP+317-326+sequence+does+not+involve+G3BP1.">Revisiting G3BP1 as a RasGAP binding protein: sensitization of tumor cells to chemotherapy by the RasGAP 317-326 sequence does not involve G3BP1.</a> PLOS ONE. 6 (12), (2011).</li><li>Kennedy, D., French, J., Guitard, E., Ru, K., Tocque, B., Mattick, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+G3BPs:+tissue+specific+expression,+chromosomal+localisation+and+rasGAP(120)+binding+studies.">Characterization of G3BPs: tissue specific expression, chromosomal localisation and rasGAP(120) binding studies.</a> Journal of Cellular Biochemistry. 84 (1), 173-187 (2001).</li><li>Kobayashi, T., Winslow, S., Sunesson, L., Hellman, U., Larsson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC&#945;+Binds+G3BP2+and+Regulates+Stress+Granule+Formation+Following+Cellular+Stress.">PKC&#945; Binds G3BP2 and Regulates Stress Granule Formation Following Cellular Stress.</a> PLOS ONE. 7 (4), (2012).</li><li>Tourri&#232;re, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RasGAP-associated+endoribonuclease+G3Bp:+selective+RNA+degradation+and+phosphorylation-dependent+localization.">RasGAP-associated endoribonuclease G3Bp: selective RNA degradation and phosphorylation-dependent localization.</a> Molecular and Cellular Biology. 21 (22), 7747-7760 (2001).</li><li>Costa, M., Ochem, A., Staub, A., Falaschi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+DNA+helicase+VIII:+a+DNA+and+RNA+helicase+corresponding+to+the+G3BP+protein,+an+element+of+the+ras+transduction+pathway.">Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway.</a> Nucleic acids research. 27 (3), 817-821 (1999).</li><li>Solomon, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+structural+features+of+caprin-1+mediate+its+interaction+with+G3BP-1+and+its+induction+of+phosphorylation+of+eukaryotic+translation+initiation+factor+2alpha,+entry+to+cytoplasmic+stress+granules,+and+selective+interaction+with+a+subset+of+mRNAs.">Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs.</a> Molecular and Cellular Biology. 27 (6), 2324-2342 (2007).</li><li>Soncini, C., Berdo, I., Draetta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ras-GAP+SH3+domain+binding+protein+(G3BP)+is+a+modulator+of+USP10,+a+novel+human+ubiquitin+specific+protease.">Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease.</a> Oncogene. 20 (29), 3869-3879 (2001).</li><li>Zekri, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+fetal+growth+and+neonatal+survival+by+the+RasGAP-associated+endoribonuclease+G3BP.">Control of fetal growth and neonatal survival by the RasGAP-associated endoribonuclease G3BP.</a> Molecular and Cellular Biology. 25 (19), 8703-8716 (2005).</li><li>Martin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+G3BP1,+the+stress+granules+assembly+factor,+results+in+abnormal+synaptic+plasticity+and+calcium+homeostasis+in+neurons.">Deficiency of G3BP1, the stress granules assembly factor, results in abnormal synaptic plasticity and calcium homeostasis in neurons.</a> Journal of neurochemistry. , (2013).</li><li>Mazroui, R., Huot, M. -. E., Tremblay, S., Filion, C., Labelle, Y., Khandjian, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+of+messenger+RNA+by+Fragile+X+Mental+Retardation+protein+into+cytoplasmic+granules+induces+translation+repression.">Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression.</a> Human molecular genetics. 11 (24), 3007-3017 (2002).</li><li>Colombrita, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+is+recruited+to+stress+granules+in+conditions+of+oxidative+insult.">TDP-43 is recruited to stress granules in conditions of oxidative insult.</a> Journal of neurochemistry. 111 (4), 1051-1061 (2009).</li><li>Thomas, M. G., Tosar, L. J. M., Desbats, M. A., Leishman, C. C., Boccaccio, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Staufen+1+is+recruited+to+stress+granules+and+impairs+their+assembly.">Mammalian Staufen 1 is recruited to stress granules and impairs their assembly.</a> Journal of Cell Science. 122 (4), 563-573 (2009).</li><li>Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Barres, B. A., Hell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NS21:+re-defined+and+modified+supplement+B27+for+neuronal+cultures.">NS21: re-defined and modified supplement B27 for neuronal cultures.</a> Journal of neuroscience. 171 (2), 239-247 (2008).</li><li>Xia, Z., Dudek, H., Miranti, C. K., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Influx+via+the+NMDA+Receptor+Induces+Immediate+Early+Gene+Transcription+by+a+MAP+Kinase/ERK-Dependent+Mechanism.">Calcium Influx via the NMDA Receptor Induces Immediate Early Gene Transcription by a MAP Kinase/ERK-Dependent Mechanism.</a> The Journal of Neuroscience. 16 (17), 5425-5436 (1996).</li><li>Dudek, H., Ghosh, A., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+transfection+of+DNA+into+neurons+in+primary+culture.">Calcium phosphate transfection of DNA into neurons in primary culture.</a> Current protocols in neuroscience. 3, (2001).</li><li>Reineke, L. C., Dougherty, J. D., Pierre, P., Lloyd, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+G3BP-induced+granules+trigger+eIF2&#945;+phosphorylation.">Large G3BP-induced granules trigger eIF2&#945; phosphorylation.</a> Molecular biology of the cell. 23 (18), (2012).</li><li>Kedersha, N., Tisdale, S., Hickman, T., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+and+quantitative+imaging+of+mammalian+stress+granules+and+processing+bodies.">Real-time and quantitative imaging of mammalian stress granules and processing bodies.</a> Methods in enzymology. 448, 521-552 (2008).</li></ol>

The authors declare that they have no competing financial interests.

The most critical steps in the protocol concern the transfection and more particularly the time lapse acquisitions, which have to be carefully monitored in order to lower down the cytotoxicity.
Culture of primary cells is not a difficult part, as long as sterile conditions are maintained and caution is taken in order to prevent damage during the dissection and cell dissociation steps. MEFs can be kept frozen at early passages. Neuron transfection with calcium phosphate has been shown to work w...

Stress granules (SGs) are non-membranous cytoplasmic foci formed as a cellular protective response to environmental stress, such as elevated temperature, oxidative stress, hypoxia, osmotic shock, UV irradiation, glucose deprivation, or viral infection1. They can be induced chemically by treatment with compounds like sodium arsenite, which triggers oxidative stress. SGs accumulate stalled translation arrested messenger ribonucleoprotein (mRNPs) complexes2, sequestering mRNAs from the translational machinery, and their assembly can be triggered by the phosphorylation of eIF2&#945; (eukaryotic initiation factor 2 &#945;). SGs are dynamic structures which exchange components with the polysomes and other granules like the P-bodies. They constitute a &#34;triage center&#34; where mRNAs are sorted and processed for either translation, reinitiation, degradation, or packaging into stable non-polysomal mRNPs3. The assembly of SGs is fast but it is a gradual process with initial numerous small aggregates which coalesce into larger granules. The use of chemical inhibitors that disrupt or stabilize microtubules shows that the microtubule network is required for SG dynamics including assembly, coalescence and disassembly processes.
The dynamic assembly of SGs is also promoted by aggregation of specific RNA-binding proteins (RNA-BP) like TIA-1 (T-cell internal antigen-1) and TIAR (TIA-1-related protein), which are able to dimerize and promote polysome disassembly and the routing of mRNAs into SGs4. G3BP (RasGAP SH3 domain binding protein) is such an RNA-BP that localizes to SGs when cells are stressed with arsenite or high temperature, and overexpression of dephosphorylated G3BP can induce SGs assembly5.
G3BP is an evolutionarily conserved RNA-BP that was initially characterized through its interaction with a Ras-GTPase activating protein (RasGAP p120 6); but this interaction was recently revisited7. The G3BP family includes two members in mammals, G3BP1 (referred to as G3BP) and G3BP2&#160;8. Both proteins colocalize in SGs, when cells are subjected to stress9. G3BPs comprise an N-terminal NTF2 domain suggested to influence their localization and oligomerization, followed by proline rich (PxxP) motifs, then C-terminal motifs associated with RNA binding: the canonical RNA-Recognition Motif (RRM) with conserved RNP1 and RNP2 motifs, followed by an arginine-glycine rich (RGG) box. Interestingly, the analysis of different parts of the protein by constructing different domains fused to EGFP showed that the NTF2-like domain and the RNA-binding domain were the most efficiently recruited to SGs, suggesting the importance of the properties of dimerization and RNA binding in the assembly of SGs. Diverse models have revealed different functions of G3BP proteins in vitro10-13. Disruption of G3BP in mice have shown the importance of this protein in developmental growth and survival14, as well as an important role for G3BP in the Central Nervous System (CNS), characterized by ataxia and defects in spatial working memory15. G3BP deficiency leads to altered neuronal plasticity and calcium homeostasis, establishing a direct link between SG formation and neurodegenerative diseases15. It is thus important to be able to study the dynamics of SGs in primary cells like neurons.
This protocol provides a simple way to observe the assembly of G3BP1-containing SGs in primary cells under arsenite treatment. It can be used to study SGs assembly under different conditions, for example different kinds of stresses. It can also be adapted to other granules or other constituents of SGs. Indeed, this protocol focuses on G3BP1, but there are other stress granules markers like TIA-1/R, TTP (tristetraprolin)2, FMRP (Fragile X Mental Retardation Syndrome protein)16, TDP-43 (transactive response DNA binding protein 43)17 or Staufen18. In particular, proteins like TIA-1/R are, like G3BP, nucleating RNA binding proteins that can induce SGs assembly when overexpressed, even if the different formed SGs can differ in function, regulation and associated transcripts. Transfection of fluorophore-tagged version of any of these key components or nucleators of SGs can be performed to image particular SGs assembly and dynamics.

<table><tbody><tr><td>DMEM/F12</td><td>Gibco</td><td>21331</td><td>Prewarm at 37 &amp;deg;C</td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360</td><td></td></tr><tr><td>Nonessential amino acids</td><td>Gibco</td><td>11140</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15096</td><td></td></tr><tr><td>Glass bottom B-35</td><td>Greiner</td><td>627860/627861</td><td>Treated or not (treated: increases attachment of adherent cells)</td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P2636</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966</td><td>Prewarm at 37 &amp;deg;C</td></tr><tr><td>HeBS</td><td>Sigma-Aldrich</td><td>51558</td><td></td></tr><tr><td>Neurobasal</td><td>Gibco</td><td>21103</td><td>Prewarm at 37 &amp;deg;C</td></tr><tr><td>2-mercaptoethanol</td><td>Gibco</td><td>31350</td><td></td></tr><tr><td>Forceps</td><td>Biotek</td><td>DU-110-A</td><td>Very thin, tips 0.1 mm (useful to remove meninges)</td></tr><tr><td>Curved forceps</td><td>Biotek</td><td>P-110-BUF</td><td>Very thin, tips 0.1 mm</td></tr><tr><td>Small scissors</td><td>Biotek</td><td>CM-85-BS</td><td>Can be useful to remove hippocampi</td></tr><tr><td>Polyplus transfection JetPEI reagent&amp;nbsp;</td><td>Ozyme</td><td>101-10</td><td>MEFs transfection, follow the forward protocol</td></tr><tr><td>Inverted laser scanning confocal microscope</td><td>Leica</td><td>SP5</td><td></td></tr></tbody></table>

visualization of g3bp stress granules dynamics in live primary cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granule&#160;formation is important in the response of cells to stress, permitting a cellular adaptation with stalled translation, until the stress has cleared, associated to prevention of apoptosis. This assay permits to study the SGs in primary cells, by following the localization of key SGs protein components (Figure 1). G3BP, a key factor of SGs assembly, is present rather diffusively in the cytoplasm of cultured MEFs or neurons (Figure 2A a and c</str...

Watch this Scientific Journal Video about Visualization of G3BP Stress Granules Dynamics in Live Primary Cells at JoVE.com

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

visualization-of-g3bp-stress-granules-dynamics-in-live-primary-cells

Research

JoVE Journal

Biology

18.2K Views.  Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535. Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

Video: Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Cancer Research

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Developmental Biology

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Immunology and Infection

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

Bioengineering

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.

This protocol presents a new reporter gene system and the experimental setup to detect transcription at DNA double-strand breaks with single-molecule sensitivity.

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far ...

Detection of G1 Phase Single-Stranded DNA Foci via RPA2 Immunofluorescent Staining

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair necessitates collaboration between numerous proteins, responsible for covering a broad range of tasks from recognizing and signaling the presence of a DNA lesion to physically repairing it. During this process, tracks of single-stranded DNA (ssDNA) are often created, which are eventually filled by DNA polymerases. The nature of these ssDNA tracks (in terms of both length and number), along with the polymerase recruited to fill these gaps, are repair pathway-specific. The visualization of these ssDNA tracks can help us understand the complicated dynamics of DNA repair mechanisms.
This protocol provides a detailed method for the preparation of G1 synchronized cells to measure ssDNA foci formation upon genotoxic stress. Using an easy-to-utilize immunofluorescence approach, we visualize ssDNA by staining for RPA2, a component of the heterotrimeric replication protein A complex (RPA). RPA2 binds to and stabilizes ssDNA intermediates that arise upon genotoxic stress or replication to control DNA repair and DNA damage checkpoint activation. 5-Ethynyl-2'-deoxyuridine (EdU) staining is used to visualize DNA replication to exclude any S phase cells. This protocol provides an alternative approach to the conventional, non-denaturing 5-bromo-2'-deoxyuridine (BrdU)-based assays and is better suited for the detection of ssDNA foci outside the S phase.

Author Spotlight: Visualizing Single-Stranded DNA During DNA Repair for Therapeutic Insights 

The following protocol presents the detection of single-stranded DNA foci in the G1 phase of the cell cycle utilizing cell cycle synchronization followed by RPA2 immunofluorescent staining.

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Super-Resolution Live Cell Imaging of Subcellular Structures

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle