an imaging technique to study the calcium response in bacteria-infected cells

An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells

Place the coverslip containing the Fluo-4 cells in an imaging or glass-bottom chamber. Use EM buffer to wash the sample three times to remove compounds potentially resulting from cell lysis. Then, add one milliliter of EM buffer. Place the chamber on an inverted fluorescence microscope stage, heated at 33 degrees Celsius. Select a microscopy field, and set up acquisition parameters, including exposure time and binning, if necessary, to optimize the Fluo-4 fluorescent signal.
To add bacteria to the sample, carefully remove 500 microliters of EM buffer from the chamber, and add 500 microliters of the bacterial suspension to obtain a final OD600 of 0.05. Immediately perform an acquisition, or wait 10 minutes to allow the bacteria to sediment onto the cells. At the end of the acquisition stream, acquire a phase contrast image of the selected field to visualize the bacteria contacting the cells and the membrane ruffles associated with bacterial invasion sites.
Repeat the acquisition procedure at intervals that are amenable to the duration of the image acquisitions, file savings, and selection of a new field to cover the whole process. At the end of the acquisition procedure, add to the sample, two micromolar of the calcium ionophore ionomycin to determine the maximal amplitude of the calcium signals. Acquire images every 3 to 5 seconds until the signal stabilizes, usually for less than 10 minutes.
Follow by adding the calcium chelator EGTA to a final concentration of 10 millimolar to determine the fluorescent signal in the absence of calcium. Acquire images every 3 to 5 seconds until signal stabilization, usually for less than 10 minutes. Carry out image analysis according to the text protocol.

an-imaging-technique-to-study-calcium-response-bacteria-infected

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

EoE Sub Articles

eoe sub articles

1. Preparations
<ol>
	<li>Bacteria preparation
	<ol>
		<li>Plate the bacteria&#8212;Shigella wild-type strain expressing the AfaE adhesin (M90T-AfaE)&#8212;onto a trypticase soy (TCS) agar plate containing 0.01% Congo red (CR) and incubate them for 18 h at 37 &#176;C. 
		Note: To increase their reproducibility, the Shigella plates obtained in step 1.1.1 are stored at 4 &#176;C and should be used within a week because all colonies on CR medium will eventually turn red over time.</li>
		<li>Inoculate TCS broth pre-culture by picking 3 red colonies from the streak plates. 
		Note: An inoculation with 3 colonies is performed to limit the probability of picking a single clone whose virulence plasmid has undergone a genetic recombination.</li>
		<li>Grow liquid bacterial cultures in a shaking incubator for 16 h at 37 &#176;C at 200 rpm. Add ampicillin at a final concentration of 75 mg/mL. Antibiotic concentrations should not exceed 3x the minimal inhibitory concentration, as an excess of antibiotic may lead to the loss of the large virulence plasmid. 
		Note: The maintenance of the Shigella virulence plasmid should be verified by plating the bacterial cultures on CR plates supplemented with appropriate antibiotic concentrations. The presence of white colonies indicates plasmid loss.</li>
		<li>Inoculate the TCS culture with the bacterial pre-culture at a 1:100 dilution. Incubate it at 37 &#176;C in a shaking incubator for 2 h at 200 rpm. Ensure that the optical density at 600 nm (OD600nm) is 0.2 - 0.4.</li>
		<li>Centrifuge the bacterial culture for 2 min at 13,000 x g at 21 &#176;C and resuspend the pellet in an equivalent volume of EM medium (120 mM NaCl, 7 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 5 mM glucose, and 25 mM HEPES, pH = 7.3).</li>
		<li>Dilute the bacterial suspension in an EM buffer at a final OD600nm of 0.1 and use it immediately or store it at 21 &#176;C and use it within the next 60 min.</li>
	</ol>
	</li>
	<li>Cell preparation
	<ol>
		<li>Culture HeLa cells in DMEM with 1 g/L of glucose supplemented with 10% fetal calf serum (FCS) and grow them at 37 &#176;C with 10% CO2. Grow TC-7 grown in DMEM with 4.5 g/L of glucose, containing 10% FCS and non-essential amino acids in a 37 &#176;C incubator containing 10% CO2.</li>
		<li>For cell maintenance, split the cells regularly to avoid a growth-contact inhibition linked to confluent states; this is critical for a high Ca2+ probe loading/transfection efficiency.</li>
		<li>Plate HeLa cells onto sterile 25-mm diameter circular coverslips in 6-well plates at a density of 3 x 105 cells/well the day before the experiment for the HeLa cells.</li>
		<li>For TC-7 cells, trypsinize and count cells. Seed them at 4 x 105 cells/well and incubate them for 5 - 7 days at 37 &#176;C in a 10% CO2 incubator before the experiments to allow for polarization.
		<ol>
			<li>Refresh the cell culture medium every day until the day of the experiment. 
			Note: Glass-bottom 35-mm diameter disposable chambers may also be used as an alternative to the coverslips-containing wells. If the latter are used, temperature control through a heated stage or objective is not sufficient, and a thermostat incubation chamber mounted on the microscope stage is required.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>On the day of the experiment, remove the medium and wash the cells 3x with 1 mL EM medium at 21 &#176;C.</li>
	<li>Load the cells with 3 mM Fluo-4/acetoxymethyl ester (Fluo-4-AM) in an EM buffer for 30 min at 21 &#176;C. 
	Note: While the loading of sub-confluent HeLa cells does not raise major problems, the loading of polarized TC-7 cells is often heterogeneous and poorly efficient. For these cells, we found that the loading efficiency is enhanced by the addition of the dispersing agent&#8212;pluronic acid&#8212;at a final concentration of 0.1% and of the anion-transporter inhibitor bromosulfophtalein at a final concentration of 20 &#181;M to the Fluo-4-AM-containing EM. The use of ratiometric chemical probes or genetically encoded FRET probes requiring dual acquisition is not compatible with the speed of acquisition required for the imaging of local Ca2+ responses.</li>
	<li>Wash the samples 3x with EM buffer and further incubate them in 1 mL of EM buffer at 21 &#176;C to allow for the hydrolysis of the AM moiety. Use Fluo-4 loaded cells immediately or within the next 90 minutes (maintained at 21 &#176;C).</li>
</ol>
2. Infection and Image Acquisition of Local Ca2+ Responses
<ol>
	<li>Place the coverslip containing the Fluo-4 loaded cells in an imaging chamber or glass-bottom chamber. Wash the samples in the imaging chamber or glass-bottom disposable chamber 3x with EM buffer to remove compounds potentially resulting from cell lysis. Add 1 mL of EM buffer.</li>
	<li>Place the chamber on an inverted fluorescence microscope stage heated at 33 &#176;C. 
	Note: This temperature is selected as a compromise between the optimal temperatures compatible with the Shigella invasion process and the slowing down of Ca2+ responses to visualize local responses. We use a 63X immersion oil objective (NA = 1.25) equipped with phase contrast rings.</li>
	<li>Select a microscopy field and set up acquisition parameters, including exposure time and binning if necessary, to optimize the Fluo-4 fluorescent signal. 
	Note: The major challenges of local Ca2+ imaging are the low amplitude and the small duration of the responses, requiring acquisition at least every 30 ms. Several commercially available back-illuminated electron-multiplying charge-coupled devices (EM-CCD) or complementary metal-oxide semiconductor (C-MOS) cameras present the required sensitivity to detect local Ca2+ responses using Fluo-4. Detection sensitivity is also an important issue in limiting photodamage or artifacts such as spontaneous responses linked to the fluorescent excitation of Fluo-4 at a high frequency. Here, an LED-based illumination of 470 nm, with a 480 &#177; 40 nm band-pass excitation filter, a 505-nm dichroic filter, and a 527 &#177; 30 nm band-pass emission used at 5% of its maximal intensity combined with a 1.0 neutral density filter were used to minimize these problems under a stream of acquisitions of limited durations. An analysis of local Ca2+ signals by batches of 120 s streams was routinely performed. Under these low illumination conditions, fluorophore photobleaching was not observed during the 2 min-stream acquisitions. Successive acquisitions on the same sample can be performed, provided different fields are used. As a general rule, a first acquisition stream is performed on a control field in the absence of bacteria stimulation to ensure the absence of spontaneous responses. If spontaneous responses are observed, the samples are washed with EM buffer until no responses are observed.</li>
	<li>To add bacteria to the sample, remove 500 &#181;L of EM buffer from the chamber and add 500 &#181;L of the bacterial suspension prepared in step 1.1.6 to obtain a final OD600nm of 0.05, with the appropriate care to avoid moving the selected microscopy field; the volumes ensure a proper mixing of the bacteria and a homogeneous distribution of the bacteria onto the cell samples.</li>
	<li>Perform an acquisition upon the bacterial addition. Alternatively, perform an acquisition 10 min following the bacterial addition, which corresponds to the time required for bacteria to sediment onto cells under the conditions used. At the end of the acquisition stream, acquire a phase-contrast image of the selected field to visualize the bacteria contacting the cells and the membrane ruffles associated with bacterial invasion sites.</li>
	<li>Repeat the acquisition procedure as in step 2.5, at intervals that are amenable to the duration of the image acquisitions, file savings, and selection of a new field to cover the whole process. 
	Note: For Shigella, we found that acquisition streams every 5 min for 20 min, following the 10 min bacterial sedimentation, allowed to cover the majority of the invasion events.</li>
	<li>At the end of the acquisition procedure, add a final concentration of 2 &#181;M of the Ca2+ ionophore ionomycin samples to determine the maximal amplitude of the Ca2+ signals. Acquire images every 3 - 5 s until signal stabilization, usually for less than 10 min.</li>
	<li>Follow by adding Ca2+ chelator ethylene glycol-bis(&#946;-aminoethyl ether)-N, N, N&#8242;, N&#8242;-tetraacetic acid (EGTA) at a final concentration of 10 mM to determine the fluorescence signal in the absence of Ca2+. Acquire images every 3 - 5 s until signal stabilization, usually for less than 10 min. 
	Note: Because of the relatively slow dynamics of the global Ca2+ variations, perform these acquisitions every 3 - 5 s (not in streaming mode), allowing for Ca2+ imaging on the same microscopic field during the successive treatments.</li>
</ol>
3. Analysis
<ol>
	<li>Express cytosolic Ca2+ variations over time as the percentage of &#916;F/F0, where &#916;F represents the variation of the average fluorescence intensity of the region of interest (ROI) and F0 the baseline fluorescence in the same ROI, to which a non-relevant region of the field devoid of cells is subtracted.
	<ol>
		<li>Remove the basal fluorescence levels for each cell in different fields, corresponding to baseline levels; these levels can be unambiguously determined due to the low frequency and relatively short duration of local Ca2+ responses in a given stream acquisition. 
		&#8203;Note: Quantify striking features of the responses related to the questions asked and the pathogenic model studied. Classically, various parameters are taken into account to analyze Ca2+ signals, including the frequency of cells showing local or global Ca2+ responses, as well as the duration, amplitude, and frequency of these responses. In the case of Shigella and local responses, another important aspect of the analysis is the spatial association of the responses with bacterial invasion sites.</li>
	</ol>
	</li>
	<li>To quantify the percentage of responsive cells showing global or local responses, draw ROIs in cells, in time series corresponding to the variations of Fluo-4 intensity.
	<ol>
		<li>Set strict parameters to identify local Ca2+ responses, such as an amplitude of three-fold above background variations of the average fluorescence intensity cell baseline, or a duration of at least 200 ms, to avoid scoring a false positive. 
		Note: As a general rule, even small local Ca2+ responses can be distinguished from any background noise by their profile. The ROIs should be large enough to integrate enough fluorescence signals to allow for the detection of local variations. Depending on the intensity of the Fluo-4 detection, these ROIs may correspond to circles with diameters ranging from 2 - 5 mM.</li>
		<li>Measure the variations of the average Fluo-4 fluorescence intensity in 2 regions in a distinct area of the same cell, to determine whether the responses are local or global. 
		Note: The analysis of a large number of cells is facilitated by the use of imaging software permitting the online screen display of the variations of the average fluorescence intensity over time for the selected regions. Commercially available imaging software has such an option, but this can also be performed using the Icy freeware, using the ROI Intensity Evolution plugin.</li>
		<li>Determine the percentage of cells showing local responses. 
		Note: This percentage is calculated from the total number of cells analyzed in the microscopy field, which should be in sufficient numbers to support a statistical significance using a non-parametric test.</li>
		<li>Determine the duration of the local responses. 
		Note: Local Ca2+ responses can be further distinguished according to their duration ranges (i.e., less than 500 ms, between 5 and 10 s, or above 10 s) and their association with Shigella invasion sites.</li>
	</ol>
	</li>
	<li>Quantify the amplitude of the responses. First, calibrate the maximal Ca2+ and cell background levels determined in step 2.1.7. Since the Fluo-4 load may vary for each cell, perform these determinations for individual cells in the field.
	<ol>
		<li>Express the amplitude of the responses as a relative percentage of the maximal response.</li>
		<li>Perform statistical testing using a normality test, followed by a parametric test to analyze potential differences in the amplitude of responses between samples.</li>
		<li>Determine the association of these responses relative to the bacterial invasion sites by their respective localization to the bacterial-induced membrane ruffles visualized in the corresponding phase contrast images.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Metamorph version 7.7</td>
			<td>Universal Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CoolLED illumination system pE-2</td>
			<td>Roper Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dish 35 mm, high</td>
			<td>IBIDI</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypticase Soy (TCS) broth</td>
			<td>Thermofisher</td>
			<td>B11768</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS agar</td>
			<td>Thermofisher</td>
			<td>B11043</td>
			<td></td>
		</tr>
		<tr>
			<td>Congo red</td>
			<td>Sigma-Aldrich</td>
			<td>75768</td>
			<td></td>
		</tr>
		<tr>
			<td>M90T-AfaE</td>
			<td>Sun et al. 2017</td>
			<td></td>
			<td>Shigella flexneri serotype V. expressing the AfaE adhesin</td>
		</tr>
		<tr>
			<td>ipgD-AfaE</td>
			<td>Sun et al. 2017</td>
			<td></td>
			<td>Isogenic ipgD mutant strain expressing the AfaE adhesin</td>
		</tr>
	</tbody>
</table>

Source: Smail, Y., et al. <a href="https://app.jove.com/v/57728">Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for detecting local and global calcium ion response upon Shigella infection of human epithelial cells. Shigella invades host cells and releases invasion effectors, impacting intracellular calcium levels. The amplitude and frequency of the local and global cytosolic calcium are studied using a fluorescent indicator.

1. Preparations

	Bacteria preparation
	
		Plate the bacteria&#8212;Shigella wild-type strain expressing the AfaE adhesin (M90T-AfaE)&#8212;onto a ...

Take an imaging chamber containing a culture of human epithelial cells loaded with a fluorescent indicator of intracellular calcium.
Add a growth medium containing calcium ions. Introduce Shigella, a pathogenic bacterium, and incubate.
The bacterial secretion system penetrates the host cell and releases invasion effectors. Invasion triggers actin rearrangement, forming membrane ruffling around bacteria.
At the invasion site, phospholipase C, or PLC, on the plasma membrane is activated, and inositol triphosphate receptors cluster on the endoplasmic reticulum or ER.
PLC hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate to inositol triphosphate, or IP3, that accumulates locally due to dense actin meshwork.
IP3 binds to receptors on the ER, causing calcium release. Calcium indicators bind to released ions and emit fluorescence, displaying a local calcium response.
IP3 slowly diffuses through the actin, further depleting the ER calcium level and activating a plasma membrane calcium channel, leading to calcium influx .
Fluorescence across the cell indicates delayed global calcium response.

28 ビュー. 細菌感染細胞のカルシウム応答を研究するイメージング技術

ビデオ: 細菌感染細胞のカルシウム応答を研究するイメージング技術 - 実験

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

多パラメータ光流体プラットフォームを用いたカルシウム流入とHIV-1感染の単細胞特性

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

JoVE Journal

免疫・感染学

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

生体 内 カルシウムイメージングのための定位ウイルス注入および勾配指数レンズ移植

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

神経科学

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

マウス膵島細胞の亜集団におけるカルシウムダイナミクスのイメージング

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells

記録

An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells