The authors would like to thank the patients for their informed consent and participation in the research study, and the clinical personnel for their excellent assistance. Figure 1A was created with BioRender.com.&#160;This work was supported by grants from Science Foundation Ireland-namely a career development award (CDA) to K.N. (SFI-13/CDA/2171), a research centre grant (SFI-12/RC/2273), and a research centre spoke award (SFI-14/SP/2710) to APC Microbiome Ireland. P.F. also received funding from SFI/20/RP/9007.

Quantifying Cell Death in Human Colonoids in Response to Cytotoxic Perturbagens

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K.N. was in receipt of research funding from AbbVie Inc. during the time the work was completed. This funding was in the context of a research centre spoke award (SFI-14/SP/2710) to APC Microbiome Ireland.

Several methods have been developed for quantitative analyses of cell death in intestinal organoids. Examining the disruption of intestinal organoid morphology by light microscopy is a straightforward approach to quantifying the effects of cytotoxic substances11. However, morphological changes are not a direct measurement of cell death, and the method is only semiquantitative. Another method is to evaluate organoid metabolic activity using an MTT or ATP assay10,11. It is important to note that these assays can only determine changes in cell viability and must be validated with a cell death assay. Other fluorometric cell death assays using DNA binding dyes have been reported12,13. A non-imaging approach using a fluorescent microplate reader is possible and allows for high throughput12. However, this method measures the average signal of an entire well, making it unsuitable for heterogeneous populations. It also requires the use of a microplate reader with Z-height adjustment. Fluorescent imaging-based techniques can be used for single-organoid analysis and capture cellular/subcellular and morphological data. Automated confocal high-content imaging (HCI) systems can generate large amounts of data at a high throughput13. Unfortunately, confocal HCI needs specialized equipment, uses complex protocols, typically requires commercial image analysis software, and is expensive.
Our protocol for quantitative analysis of colonoid cell death at multiple time points is straightforward, simple, and inexpensive. However, compared to automated HCI and plate reader systems, it is time-consuming and has reduced throughput. Another limitation of our method is the use of widefield as opposed to confocal microscopy. Confocal microscopy is more suitable for imaging thick 3D samples such as organoids as it reduces out-of-focus signal and can acquire serial optical sections (Z-stacks). However, confocal imaging typically requires longer acquisition times and high-intensity lasers that increase phototoxicity/photobleaching. It is critical to note that fluorescent cell death dyes like SYTOX Green are only suitable for measuring forms of cell death where there is loss of cell membrane integrity such as necrosis, late apoptosis-associated secondary necrosis, necroptosis, and pyroptosis21. There are some forms of regulated cell death where the cell membrane remains impermeable at least during the early phases of cell death, such as caspase-dependent apoptosis. However, this protocol could be easily modified to also incorporate imaging of a caspase 3/7 activity fluorescent reporter22. This would provide additional data to help characterize the specific cell death modality.
We used our protocol to demonstrate the cytotoxic synergistic interaction between the cytokines IFN-&#947; and TNF-&#945; (Figure 2C), which we have previously reported in CD patient-derived organoids9,10. The physiological relevance of this form of synergism has also been demonstrated in murine models of hemophagocytic lymphohistiocytosis and sepsis23. Several mathematical reference models and approaches have been implemented for quantifying synergy between combinations of biological agents24,25. They differ in terms of their complexity, the number of factors they consider, and the threshold for considering an interaction to be synergistic24,25. Some models need prior knowledge of the biological agents tested, make certain assumptions about the activity of agents, and can require comprehensive dose-response curves for each single and combination treatment25. The method we selected to measure synergy is a modification of the coefficient of drug interaction (CDI) model, which has previously been used to measure the inhibitory effects of chemotherapy drug combinations on cancer cell line proliferation26. The CDI is a Bliss independence model; when calculating the predicted combined effect of two perturbagens Bliss independence assumes that they target separate pathways and have independent mechanisms of action27. For an interaction between perturbagens to be synergistic the actual combined effect must be greater than the predicted effect. This model is appropriate for our experimental setup as IFN-&#947; and TNF-&#945; are known to have different receptors and downstream signaling components. Further, Bliss independence allows for the calculation of a coefficient of interaction to quantify synergism and does not require dose-response datasets.
There are a few key factors that must be considered to ensure optimal results for this protocol. It is important that colonoids are propagated to a high density (Figure 1Bi), that they are approximately 25-50 &#181;m in diameter, and are actively proliferating before attempting to seed cells. The use of suboptimal cultures of colonoids for assays may result in insufficient cell numbers, low colonoid recovery, and inconsistent experiments. For reproducible results, it is also important to seed the density of colonoids consistently between experiments. It has previously been demonstrated that the in vitro response to inflammatory cytokines can be influenced by cell seeding density28,29. Another common issue is the formation of air bubbles in the BME dome, which can affect imaging. This can be prevented by using the reverse pipetting technique. This technique also results in more consistent seeding.
Further, if imaging multiple time points, prepare a Max Toxicity condition for each time point. Triton-X 100, a nonionic surfactant, is commonly used as a positive control (Max Toxicity condition) for cytotoxicity assays. The addition of Triton-X 100 lyses and kills the colonoids, allowing the fluorescent cell death dye to enter the cells. Using a Max Toxicity condition from an earlier time point will result in inaccurate and inconsistent normalization of data due to the fluorescent signal decaying over time.
A final point to consider is the choice of BME used for colonoid culture. There are several commercial producers of BME; however, for our protocol, we have only tested the brand included in the Table of Materials. A recent study using patient-derived pancreatic cancer organoids found that the commercial source of BME altered cell proliferation rates but had no significant effect on response to chemotherapy drugs or gene expression30. With this in mind, we expect the trend of results to be similar between BME brands for our protocol, but we recommend using the same brand consistently.
We demonstrated how this protocol can be used for the analysis of IFN-&#947; and TNF-&#945; induced cell death using CD patient-derived colonoids. Patient-derived intestinal organoids are a powerful tool to study CD as they retain many characteristics of the disease, including increased sensitivity to the cytotoxic effects of TNF-&#945;31. However, the protocol could be easily modified to investigate cytotoxic effects of perturbagens other than cytokines or disease states other than IBD such as colorectal cancer (we have successfully tested the protocol using non-IBD colonoids). We believe this method is useful for any research area concerned with cell death mechanisms, epithelial barrier function, or intestinal mucosal immunology.

The intestinal epithelium creates a physical semipermeable barrier between the contents of the gut lumen and the underlying tissues. To maintain this barrier effectively, intestinal epithelial cells (IECs) undergo an extremely high cellular turnover, with a continuous cycle of cell death and regeneration. However, during inflammatory disorders, such as inflammatory bowel disease (IBD), higher levels of aberrant cell death occur1. This may promote a breakdown in barrier function and activation of the immune system, triggering further inflammation. In Crohn&#39;s disease (CD), a form of IBD, it has been shown that cytokine signaling contributes to the increased levels of IEC death2. By studying how cytokine signaling induces cell death of IECs, it is hoped that improved treatments can be developed for patients with IBD and other intestinal inflammatory disorders1.
In biology and drug target discovery research, synergy is generally understood to occur when a biological system treated with combinations of individual stimuli shows a response to the combination that is greater than the combined additive effects of the single stimuli alone. Synergistic interactions between cytokines have been well documented in driving innate antiviral responses3. Cytokines have also been known to induce cell death synergistically, including in IECs4. However, the role synergistic cytotoxic cytokine signaling plays in intestinal inflammatory disorders such as IBD is understudied.
Human intestinal organoids are 3D microtissues produced in vitro that are generated from intestinal epithelial stem cells. Intestinal organoids can be grown from gut mucosal biopsies obtained from patients with IBD and retain many characteristics of the disease5,6. Organoids have proven to be an ideal model system for studying cytokine cytotoxicity in the context of intestinal inflammation7,8. Previously, our group has characterized the synergistic killing effects of the IBD-relevant cytokines IFN-&#947; and TNF-&#945; in CD patient-derived colonic organoids (colonoids)9,10. However, the exact mechanisms involved in mediating this form of synergistic cell death remain elusive. There are also potentially many more uncharacterized cytotoxic cytokine interactions that are relevant to intestinal inflammatory disorders.
Several protocols are available to study cell death in intestinal organoids10,11,12,13; however, they each have drawbacks. Some of these techniques only measure cell viability and do not measure cell death directly, are incapable of evaluating single-organoid responses, or require expensive equipment and complex protocols. Robust and straightforward methodologies are needed to quantify organoid cell death and perturbagen interactions in intestinal organoids. The protocol we present is a simple and inexpensive approach to measure single-organoid responses to cytotoxic cytokines but can be used for any type of stimulus or perturbagen. We also demonstrate how to use the Bliss independence synergy model to calculate a coefficient of perturbagen interaction (CPI) that describes cytotoxic cytokine interactions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B Solution</td>
			<td>Sigma-Merck</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>A-83-01</td>
			<td>Sigma-Merck</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRender</td>
			<td>Science Suite Inc.</td>
			<td>N/A</td>
			<td>Scientific illustration software</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Merck</td>
			<td>A2058</td>
			<td>Essentially IgG-free, low endotoxin</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>Sigma-Merck</td>
			<td>SML1046</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 48-well Clear TC-treated Multiple Well Plates, Individually Wrapped, Sterile</td>
			<td>Corning</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Basement Membrane Extract, Type 2, Pathclear</td>
			<td>R&#38;D Systems</td>
			<td>3532-010-02</td>
			<td>Basement membrane extract</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Merck</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline</td>
			<td>Sigma-Merck</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL Digital Inverted Fluorescence Microscope</td>
			<td>Invitrogen</td>
			<td>AMF4300</td>
			<td>Digital inverted epifluorescence microscope</td>
		</tr>
		<tr>
			<td>EVOS 40x Objective, fluorite, LWD, phase-contrast</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEP4683</td>
			<td>Long working distance 40x fluorescence objective</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ (Windows version)</td>
			<td>Open-source software</td>
			<td>N/A</td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Sigma-Merck</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin Solution</td>
			<td>Sigma-Merck</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>STEMCELL Technologies</td>
			<td>100-0485</td>
			<td>Enzyme-free cell dissociation reagent</td>
		</tr>
		<tr>
			<td>GlutaMAX-1</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-alanyl-L-glutamine dipeptide supplement</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5 (Windows version)</td>
			<td>Dotmatics</td>
			<td>N/A</td>
			<td>Data graphics and statistics software</td>
		</tr>
		<tr>
			<td>Greiner 15 mL Polypropylene Centrifuge Tube, Sterile with conical bottom &#38; Screw Cap</td>
			<td>Cruinn</td>
			<td>188261CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant EGF (animal free)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Merck</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Merck</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>InvivoGen</td>
			<td>ant-nr-05</td>
			<td>Broad range antimicrobial reagent</td>
		</tr>
		<tr>
			<td>Nunc Edge 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td>
			<td>ThermoFisher Scientific</td>
			<td>15543115</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IFN-gamma Protein</td>
			<td>R&#38;D Systems</td>
			<td>285-IF</td>
			<td>Resuspend in sterile filtered 0.1% PBS/BSA</td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha Protein</td>
			<td>R&#38;D Systems</td>
			<td>210-TA</td>
			<td>Resuspend in sterile filtered 0.1% PBS/BSA</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Merck</td>
			<td>S7067</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Cap Low Retention Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>3451</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Green Nucleic Acid Stain - 5 mM Solution in DMSO</td>
			<td>Invitrogen</td>
			<td>S7020</td>
			<td>Fluorescent cell death dye, protect from light</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Merck</td>
			<td>93420</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma-Merck</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryple Express</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td>Enzymatic dissociation reagent</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>MedChemExpress</td>
			<td>HY-10071</td>
			<td>Inhibitor of ROCK-I and ROCK-II</td>
		</tr>
	</tbody>
</table>

quantification of cytokine-induced cell death in human colonic organoids using live fluorescence microscopy

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-&#945; and IFN-&#947;, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

Using this protocol, we demonstrated how CD patient colonoids can be used to study the cytotoxic effects of the IBD-relevant cytokines IFN-&#947; and TNF-&#945; on primary epithelium. We used a commercially available fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), which can only enter cells that have a compromised cell membrane where it is then activated by binding to nucleic acids. We co-treated colonoids with cytokines and the fluorescent cell death dye and performed live cell imaging at 8 h and 24 h with an inverted epifluorescence&#160;microscope. Representative transmission/fluorescent overlay images at 8 h indicate that only the IFN-&#947; + TNF-&#945;-treated colonoids are positive for fluorescent signal; however, there are only a small number of fluorescent cells (Figure 2A). Cell blebbing, a morphological indicator of cell death20, can also be observed in the IFN-&#947; + TNF-&#945; condition. At 24 h, colonoids treated with IFN-&#947; + TNF-&#945; display large regions positive for fluorescent signal (Figure 2A). There is also a clear breakdown in the morphology of the colonoid-the central lumen is no longer visible, and the epithelial barrier has been completely disrupted.
To quantify the cell death dye signal, we used open-source image analysis software to calculate the fluorescent intensity of each colonoid. We then normalized the data by expressing the mean of each condition as a percentage of the Max Toxicity treatment. At 8 h, homeostatic or background cell death in the BSA control colonoids was relatively low (7.7% of Max Toxicity) (Figure 2B). There were no statistically significant changes in cell death levels at this time point; however, conditions treated with TNF-&#945; displayed a small increase in cytotoxicity (Figure 2B). After 24 h, cell death levels had increased for all cytokine-treated conditions. However, there was minimal change in cell death for the BSA Control condition between time points (7.5% of Max Toxicity at 24 h). The colonoids treated with IFN-&#947; + TNF-&#945; had the largest increase in cell death levels compared to BSA control (29.4% of Max Toxicity). The difference in cell death levels between the combined treatment and the single cytokine treatments (IFN-&#947;, TNF-&#945;) was highly significant. These results suggest the possibility of a cytotoxic synergistic interaction between IFN-&#947; and TNF-&#945; at 24 h.
We used the CPI to quantify the cytotoxic interactions between cytokine treatments and to determine if they were synergistic. Interactions between cytokines are considered synergistic when the CPI value is &#60;1, additive when =1, or antagonistic when &#62;1. We calculated CPI values per time point (Figure 2C). At 8 h, the CPI value indicated slight synergism (0.99), with the CPI value decreasing substantially at 24 h (0.83). This analysis confirmed that the interaction between IFN-&#947; and TNF-&#945; at 24 h was synergistic. Further, it illustrates how in this context synergism between IFN-&#947; and TNF-&#945; is time-dependent.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66759/66759fig01v2.jpg" /> 
Figure 1: Schematic of experimental workflow and troubleshooting. (A) Schematic overview of protocol. (B) Representative images. (Bi) Light microscopy image illustrating the optimal density of the culture and optimal size of colonoids prior to passaging for an assay. Scale bar = 500 &#181;m. (Bii) Light microscopy image illustrating optimal size of colonoid fragments after dissociation; fragments highlighted in red. Scale bar = 100 &#181;m. (Biii) Light microscopy image of necrotic colonoid morphology after MT treatment (with Triton X-100). Scale bar = 25 &#181;m. (Biv) Light microscopy image of two colonoids overlapping in the same focal plane. Scale bar = 25 &#181;m. (Bv) Light microscopy image of colonoid cell debris present after passaging; debris highlighted in red. Scale bar = 25 &#181;m. Abbreviations: ROI = region of interest; MFI = mean fluorescence intensity; MT = Max Toxicity. <a href="https://www.jove.com/files/ftp_upload/66759/66759fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66759/66759fig02v2.jpg" /> 
Figure 2: Quantitative analysis of cytokine-induced cell death in human CD colonoids. (A) Representative live microscopy images of CD colonoids treated with SYTOX Green Nucleic Acid Stain (fluorescent cell death dye) and cytokines at 8 h and 24 h; transmission and GFP (green color) channels overlayed. Colonoids were treated as follows: 1) PBS/BSA, 2) 10 ng/mL IFN-&#947;, 3) 10 ng/mL TNF-&#945;, 4) 10 ng/mL IFN-&#947; + 10 ng/mL TNF-&#945;. Scale bars = 25 &#181;m. (B) Quantitative analysis of CD colonoids treated with the fluorescent cell death dye and cytokines at 8 and 24 h; data are expressed as a % of the MT condition. N = 2 CD colonoid lines, 11-16 colonoids imaged per condition. (C) CPI calculated per time point using the dataset from B, N = 2 CD colonoid lines. Data are expressed as means &#177; SE. In B, two-way ANOVA analysis was performed followed by Bonferroni post-tests, *P &#60; 0.05, ***P &#60; 0.001 as indicated. Abbreviations: CD = Crohn&#39;s disease; GFP = green fluorescent protein; CPI = coefficient of perturbagen interaction; PBS = phosphate-buffered saline; BSA = bovine serum albumin; TNF-&#945; = tumor necrosis factor-alfa; IFN-&#947; = interferon-gamma; MT = Max Toxicity. <a href="https://www.jove.com/files/ftp_upload/66759/66759fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of culture media for protocol. To prepare Organoid Proliferation Media, combine L-WRN conditioned media and Serum-free Media 1:1, then add supplements. Organoid Proliferation Media should be used within 2 weeks of preparation. Please note all complete media should be stored at 4 &#176;C. <a href="https://www.jove.com/files/ftp_upload/66759/66759_Table 1 (1).xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Experimental 96-well plate layout and cytokine treatments. <a href="https://www.jove.com/files/ftp_upload/66759/66759_Table 2 (1).xlsx" target="_blank">Please click here to download this Table.</a>

Author Spotlight: Understanding Cytokine-Induced Cell Death in Intestinal Epithelial Cells Using Human Organoids

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ...

quantification-cytokine-induced-cell-death-human-colonic-organoids

Research

JoVE Journal

Biology

1.2K Views.  University College Cork. This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Video: Author Spotlight: Understanding Cytokine-Induced Cell Death in Intestinal Epithelial Cells Using Human Organoids

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...