This work was supported by the Ministry of Defence of the Czech Republic -&#160;Long-term organization development plan Medical Aspects of Weapons of Mass Destruction of the Faculty of Military Health Sciences, University of Defence; the Ministry of Education, Youth and Sport, Czech Republic (Specific Research Project No: SV/ FVZ201703) and PROGRES Q40/06. Thanks also to Daniel Diaz for his kind assistance in English language revision.

1. Preparation of culture media, solutions, and dishes
<ol>
	<li>Autoclave the coverslips (22 x 22 mm). Prepare 6 well plates. Thaw fetal bovine serum (FBS) and antibiotic penicillin/streptomycin and warm the culture medium to room temperature (RT). Use trypsin-EDTA (0.25%) and 1x PBS (phosphate buffered saline with calcium and magnesium) to passage the cells.</li>
	<li>Prepare fresh 4% paraformaldehyde (PFA) in dH2O (800 mg of PFA in 20 mL of dH2O). The PFA must be freshly prepared for each experiment. Stir and heat the solution at 55 &#176;C for 30 min in the hood. Cool down at RT. Add 1 M sodium hydroxide until the solution becomes clear (pH = 7.2&#8211;7.4). Store at 4 &#176;C for up to 1 week. 
	Note: PFA is toxic; always wear adequate personal protective equipment and prepare in the chemical hood.</li>
	<li>Prepare 500 mL of culture media, DMEM (Dulbecco&#180;s Modified Eagle&#180;s medium) containing 10% FBS, 1% penicillin/streptomycin, and 2% glutamine.</li>
	<li>Prepare 13 mL of 1% gelatin solution in sterile dH2O (130 mg of gelatin in 13 mL of dH2O). Use 2 mL of 1% gelatin for each well in a 6 well plate. Keep sterile.</li>
	<li>Clean the laminar flow hood using 70% ethanol. Place the required material inside the laminar flow hood before starting the experiment.</li>
</ol>
2. Cell culture for immunocytochemistry staining
<ol>
	<li>Thaw the cells (in this study C2C12, MEF, NHLF, and skin fibroblasts) using standard techniques and plate them in a T75 flask supplemented with ~10&#8211;12 mL of the prepared media. Incubate at 37 &#176;C/5% CO2/90% relative humidity (RH) until the cells reach 70% confluence.</li>
	<li>Remove the cells from the incubator and place them in the laminar flow hood. Remove the culture media and rinse the cells briefly 2x with 1x PBS. Add ~2 mL of 0.25% trypsin-EDTA into the T75 flask and incubate at 37 &#176;C for ~5 min. Check periodically on the inverted microscope to monitor cell detachment. 
	NOTE: The incubation time depends on the cell line and therefore must be determined empirically.</li>
	<li>Gently resuspend the cells in 10 mL of culture media, pipetting carefully to create a single cell suspension. Rinse the flask again if necessary.</li>
	<li>Place the cell suspension in a 50 mL conical tube and centrifuge for 5 min at ~200 x g. Decant the supernatant, add 10 mL of culture media, and gently resuspend the pellet. Take 20 &#181;L of the cell suspension and mix in a 1:1 ratio with trypan blue and count in a cytometer following the standard method.</li>
	<li>Place one coverslip inside each well of a 6 well plate using tweezers. Coat the coverslips with gelatin by pouring ~2 mL into the wells. This will help the cells attach to the coverslips. Remove the gelatin solution and let air-dry for a few minutes. The coverslips are now ready for cultivation of the cells. Start the cultivation immediately.</li>
	<li>Seed 100,000 fibroblasts into each well and add 2 mL of culture media. Incubate the cells for 24 h at 37 &#176;C/5% CO2/90% RH. At this point, the cells can be treated according to the needs of the user. Treatments to induce ciliation have been previously described4,5. 
	NOTE: The initial seeding number depends on the cells&#8217; doubling time and should be determined accordingly.</li>
</ol>
3. Immunofluorescent staining of primary cilia in vitro
<ol>
	<li>Warm the 4% paraformaldehyde to RT. Prepare Pasteur pipettes, 1x PBS (RT), waste container, 15 mL conical tubes, micropipettes (0.5&#8211;10 &#181;L, 20&#8211;200 &#181;L, and 100&#8211;1,000 &#181;L) and tips. Take the cells from the incubator and place them in the bench. 
	NOTE: The staining procedure does not need to be performed in sterile conditions. All solutions must be at RT.</li>
	<li>Remove media from each well. Leave the coverslip inside the well. Very gently wash the cells 3x with 2 mL of 1x PBS. Using a Pasteur pipette, add 2 mL of 4% PFA into each well to fix the cells. Incubate for 10 min at RT. Remove the PFA and wash 3x with 1x PBS. 
	NOTE: Always use a sufficient volume to cover the entire coverslip during the incubation periods. Never let the cells dry. Never pour any of the solutions directly onto the coverslip.</li>
	<li>Prepare 0.5% Triton X-100 in 13 mL of 1x PBS 10 min before use. Add 2 mL into each well. Incubate for 15 min. Wash gently 4x with 1x PBS. 
	NOTE: Triton X-100 is insoluble in PBS at RT. Heat the 0.5% Triton X-100 solution to 37 &#176;C in a water bath to dissolve it.</li>
	<li>Thaw goat serum 5 min before use. Dilute the goat serum in 1x PBS in a 1:20 ratio as a blocking solution. Add 150 &#181;L to each coverslip and incubate for 20 min at RT. 
	NOTE: Prolong the blocking period up to 60 min if necessary. Do not wash the cells after blocking with goat serum.</li>
	<li>Thaw the primary antibodies (i.e., anti-acetylated alpha tubulin and anti-gamma tubulin) 5 min before use. Dilute the antibodies separately in 1x PBS as follows: mouse anti-acetylated alpha tubulin in a 1:800 ratio and rabbit anti-gamma tubulin in a 1:300 ratio. Remove the blocking solution. Do not wash. Add 150 &#181;L of both antibody dilutions to the coverslips and incubate for 60 min at RT. 
	NOTE: If incubating overnight use 500&#8211;1,000 &#181;L of the primary antibody solutions, seal the 6 well plate with paraffin film, and store at 4 &#176;C. Alternatively, use 150 &#181;L of antibody and incubate in a humidity chamber.</li>
	<li>Remove the primary antibodies. Wash the coverslips very gently 3x with 2 mL of 1x PBS. Prepare the secondary antibodies in 1x PBS by separately diluting Cy3 sheep anti-mouse and Alexa Fluor488 goat anti-rabbit in a 1:300 ratio. Add 150 &#181;L of both secondary antibody dilutions to the coverslips. Incubate for 45 min at RT in the dark. 
	NOTE: Incubate in the dark to avoid photobleaching. Other combinations of secondary antibodies can be used as needed.</li>
	<li>Prepare a DAPI (4&#39;, 6-diamidino-2-phenylindole) solution according to the manufacturer&#39;s instructions. Store the excess aliquots at -20 &#176;C. Dilute 10 &#181;L from a stock aliquot (1:5,000) in 50 mL of 1x PBS. Add 2 mL of this dilution to the coverslips. Incubate for 5 min at RT in the dark. 
	NOTE: It is important to incubate the cells in the dark to avoid photobleaching. The DAPI dilution can be stored at 4 &#176;C for up to 1 month.</li>
	<li>Prepare 2 needles, slides, tweezers, and mounting media. Label the slides.</li>
	<li>Remove the DAPI solution from the wells. Wash 3x with 1x PBS. Put one drop of mounting media on each slide. Use the needle to gently lift the coverslip from the well&#8217;s bottom. Flip the coverslip using the tweezers and gently place it over the drop of mounting media. Carefully remove any bubbles.</li>
	<li>Protect the slides from light and store them overnight at 4 &#176;C.</li>
	<li>Use a fluorescent or confocal microscope with high magnification to visualize the primary cilia. 
	NOTE: The slides can be stored in the dark at 4 &#176;C for up to 2 months.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cilium:+Cellular+Antenna+and+Central+Processing+Unit.">The Cilium: Cellular Antenna and Central Processing Unit.</a> Trends in Cell Biology. 27 (2), 126-140 (2017).</li><li>Morleo, M., Franco, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Autophagy-Cilia+Axis:+An+Intricate+Relationship.">The Autophagy-Cilia Axis: An Intricate Relationship.</a> Cells. 8 (8), 905 (2019).</li><li>Ott, C., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+live+primary+cilia+dynamics+using+fluorescence+microscopy.">Visualization of live primary cilia dynamics using fluorescence microscopy.</a> Current Protocols in Cell Biology. , (2012).</li><li>Sun, S., Fisher, R. L., Bowser, S. S., Pentecost, B. T., Sui, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+architecture+of+epithelial+primary+cilia.">Three-dimensional architecture of epithelial primary cilia.</a> Proceedings of the National Academy of Sciences. 116 (19), 9370-9379 (2019).</li><li>Lauring, M. 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=New+software+for+automated+cilia+detection+in+cells+(ACDC).">New software for automated cilia detection in cells (ACDC).</a> Cilia. 8 (1), 1 (2019).</li><li>Conroy, P. 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The authors have nothing to disclose.

Several authors have described diverse methods for the detection of primary cilia, sometimes also describing various fixation methods that can affect their detection6,20,21,22. Regardless, it is difficult to find a complete and straightforward protocol for detection. The ready availability of such a method would undoubtedly be of great assistance to the study of primary cilia investigation, esp...

Primary cilia are sensory, solitary, membrane-bound, nonmotile structures associated with the cell&#8217;s mother centriole. Primary cilia are found on most vertebrate cells with the exception of red blood cells, adipocytes1, and hepatocytes2. Primary cilia are formed as an elongated axoneme composed by microtubules, whose main component is &#945;-tubulin. The axoneme grows from the basal body, which is structured from &#947;-tubulin. The length of the primary cilia varies between 2&#8211;10 &#181;m; however, its dimensions can change during glycylation, starvation, hypoxia, cytotoxic stress, or after exposure to ionizing radiation3,4,5,6,7. Usually, cells have only one primary cilium, which is involved in morphogenesis and cell signalling pathways important for cell proliferation and differentiation8,9.
Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases, and resorbed before entering mitosis in a process associated with tubulin deacetylation mediated by HDAC6 (histone deacetylase 6)10. The exact moment of primary cilia resorption depends upon cell type and the expression of genes directly involved in this process, such as Aurora A, Plk1, TcTex-111,12,13. Depending on the cell type, the primary cilia express different types of receptors, ion channels, and active signalling pathways. These include the most important signalling receptors affecting proliferation and survival, EGFR, PDGFR, and FGFR. Also included are some of the signalling pathways that may affect the function of one or more organs, including Hedgehog, Notch, and Wnt. Thanks to these receptors and signalling pathways, the primary cilia also perform a chemosensory function. This function allows primary cilia to detect specific ligands for Notch, hormones, and biologically active substances such as serotonin or somatostatin. Other specific functions exhibited by primary cilia of different lengths include reaction to changes in temperature, gravity, and osmolality14.
Primary cilia can be visualized through various methods, such as live visualization, transmission electron microscopy, 3D imaging, or by software for the automatic detection of primary cilia5,15,16,17. However, these methods are highly specialized and ongoing research needs basic, fast, and easy methods for staining primary cilia in every stage of research. Described is an easy and useful method for the detection of primary cilia in cultured cells.

<table><tbody><tr><td>6-well plate</td><td>TPP</td><td>92406</td><td>Dimensions 128x86x22 mm</td></tr><tr><td>Alexa Fluor488</td><td>Jackson ImmunoResearch</td><td>111-546-047</td><td>AffiniPure F(ab&#039;)? Fragment Goat Anti-Rabbit IgG</td></tr><tr><td>Anti-Tubulin &amp;gamma;</td><td>Sigma-Aldrich</td><td>T5192</td><td>Polyclonal Rabbit anti-Mouse IgG2a</td></tr><tr><td>C2C12</td><td>ATCC</td><td>CRL-1772</td><td>Myoblast (mouse)</td></tr><tr><td>Cy3</td><td>Sigma-Aldrich</td><td>C2181</td><td>Anti-Mouse IgG (whole molecule) F(ab&amp;prime;)2 fragment&amp;ndash;Cy3 antibody produced in sheep</td></tr><tr><td>Dapi (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Dulbecco&amp;acute;s Modified Eagle&amp;acute;s medium</td><td>Thermo Scientific</td><td>11960044</td><td>High glucose, No glutamine, Gibco</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8662</td><td>With MgCl&lt;sub&gt;2&lt;/sub&gt; and CaCl&lt;sub&gt;2&lt;/sub&gt;, Sterile-filtered, Suitable for cell culture</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Scientific</td><td>16000044</td><td>Sterile-Filtered, Gibco</td></tr><tr><td>L-Glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>MEF</td><td>ATCC</td><td>SCRC-1039</td><td>Mouse embryonic fibroblast</td></tr><tr><td>Monoclonal Anti-Acetylated Tubulin</td><td>Sigma-Aldrich</td><td>T7451</td><td>Monoclonal Anti-Acetylated Tubulin antibody produced in mouse</td></tr><tr><td>NHLF</td><td>Lonza</td><td>CC-2512</td><td>Primary lung fibroblasts (human)</td></tr><tr><td>Normal Goat Serum</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127-500G</td><td>Powder</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td>10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, Sterile-Filtered</td></tr><tr><td>ProLong Diamond Antifade Mountant</td><td>Thermo Scientific</td><td>P36961</td><td></td></tr><tr><td>Skin fibroblasts</td><td>Kindly gifted from Charles University, Faculty of Medicine in Hradec Kr&amp;aacute;lov&amp;eacute;.</td><td></td><td></td></tr><tr><td>Square Cover Slips</td><td>Thermo Scientific</td><td>22X22-1.5</td><td>Borosilicate glass, 22x22mm, Square</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>11332481001</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Thermo Scientific</td><td>25200072</td><td>Sterile-Filtered, Gibco</td></tr></tbody></table>

simple detection of primary cilia by immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

The immunofluorescent staining of primary cilia is a relatively simple procedure that results in high-quality images. In these experiments, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope following the protocol described above. The primary cilium was detected using acetylated &#945;-tubulin and &#947;-tubulin. The evaluation of primary cilia can be performed on various levels and any change in this regard can be linked to exposure to ionizing radiation, c...

Watch this Scientific Journal Video about Simple Detection of Primary Cilia by Immunofluorescence at JoVE.com

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

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10.7K Views.  University of Defence. Dit protocol zorgt voor een snelle en eenvoudige detectie van primaire trilharen in gekweekte cellen, binnen een verscheidenheid van onderzoek inspanningen. De methode kan worden gebruikt in wanorde die de basale lichamen van primaire trilharen beïnvloeden. Deze procedure kan ook worden toegepast voor het bevlekken primaire trilharen in paraffine ingebed weefsel of voor andere experimenten waar fluorescentie nodig is.Begin met autoclaving 22 bij 22 millimeter afdek slips, en de voorbe...

Video: Simple Detection of Primary Cilia by Immunofluorescence

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Immunology and Infection

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Neuroscience

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Developmental Biology

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Artificial Intelligence Approaches to Assessing Primary Cilia

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.

The use of artificial intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. ...

Volumetric Imaging and Analysis of Primary Cilia in Musculoskeletal Tissue using the ARL13B-CENTRIN-2 Mouse Model

The primary cilium is a non-motile, solitary organelle assembled by most cell types. It is essential to the development and homeostasis of the skeleton and in coordinating cell responses to biochemical and mechanical cues from their surrounding environment. Due to its micrometer-scale size, the primary cilium is challenging to image in situ to characterize in tissues, in a high throughput manner and without optical biases. An understanding of its organization in its 3D environment in vivo will be key to understanding ciliary roles in physiology and disease. The ARL13B-CENTRIN-2 mouse line presents an mCherry tag on the ciliary axoneme protein ARL13B and a green fluorescent protein (GFP) tag on the centriole protein CENTRIN-2, localized to the base of the cilium. This mouse line allows for imaging of the primary cilium without the need for staining, which reduces the number of steps in image collection and circumnavigates issues with signal-to-noise. This publication describes a step-by-step protocol to image primary cilia in musculoskeletal tissues whilst preserving the fluorescent signal. It also describes a universally applicable image analysis pipeline, enabling unbiased quantification of cilia features, such as length and 3D orientation.

This method describes the exploitation of the endogenous signal in an ARL13B-CENTRIN-2 transgenic mouse model to image primary cilia in situ, in three dimensions across large areas of mineralized tissue, from tissue collection to analysis of cilia organization. This protocol may also be applied to other types of tissues.

The primary cilium is a non-motile, solitary organelle assembled by most cell types. It is essential to the development and homeostasis of the skeleton ...

Simple Detection of Primary Cilia by Immunofluorescence

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