Name: a semi-automated and reproducible biological-based method to quantify calcium deposition in vitro
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Description: Watch this Scientific Journal Video about A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro at JoVE.com

This research was funded by the European Union&#39;s Horizon 2020 research and innovation programs under the Marie Sklodowska-Curie grant agreement No 722609 and 764474, NWO ZonMw (MKMD 40-42600-98-13007). This research was supported by BioSPX. WJ-D received funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) TRR219-project ID 322900939 and project ID 403041552

1. Cell seeding, maintenance, and calcification induction
<ol>
	<li>For culturing primary cells, use a laminar airflow cabinet, gloves, and sterile equipment. Disinfect hands and workspace before and after conducting any work. Treat all primary cells and culture media as a potential biohazard, unless proven otherwise. Preferably autoclave surplus cells and media before disposal. Do not chemically inactivate and autoclave since this will liberate toxic fumes.</li>
	<li>Culture hVSMC on uncoated cell cul...

<ol>
	<li>Taylor, A. J., Bindeman, J., Feuerstein, I., Cao, F., Brazaitis, M., O'Malley, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+calcium+independently+predicts+incident+premature+coronary+heart+disease+over+measured+cardiovascular+risk+factors:+mean+three-year+outcomes+in+the+Prospective+Army+Coronary+Calcium+(PACC)+project.">Coronary calcium independently predicts incident premature coronary heart disease over measured cardiovascular risk factors: mean three-year outcomes in the Prospective Army Coronary Calcium (PACC) project.</a> Journal of the American College of Cardiology. 46 (5), 807-814 (2005).</li><li>Arad, Y., Goodman, K. J., Roth, M., Newstein, D., Guerci, A. 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L., Speer, M. Y., Scatena, M., Giachelli, C. M., Shanahan, C. 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F., Liberman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+mechanisms+of+vascular+calcification+in+aging.">Multifaceted mechanisms of vascular calcification in aging.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 39 (7), 1307-1316 (2019).</li><li>Herrmann, J., Babic, M., T&#246;lle, M., vander Giet, M., Schuchardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+models+for+studying+vascular+calcification.">Research models for studying vascular calcification.</a> International Journal of Molecular Sciences. 21 (6), 2204 (2020).</li><li>Bowler, M. A., Merryman, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+aortic+valve+calcification:+solidifying+a+system.">In vitro models of aortic valve calcification: solidifying a system.</a> Cardiovascular Pathology: The Official Journal of the Society for Cardiovascular Pathology. 24 (1), 1-10 (2015).</li><li>Gitelman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+automated+procedure+for+the+determination+of+calcium+in+biological+specimens.">An improved automated procedure for the determination of calcium in biological specimens.</a> Analytical Biochemistry. 18 (3), 521-531 (1967).</li><li>Furmanik, M., 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=Endoplasmic+reticulum+stress+mediates+vascular+smooth+muscle+cell+calcification+via+increased+release+of+Grp78+(glucose-regulated+protein,+78+kDa)-loaded+extracellular+vesicles.">Endoplasmic reticulum stress mediates vascular smooth muscle cell calcification via increased release of Grp78 (glucose-regulated protein, 78 kDa)-loaded extracellular vesicles.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 41 (2), 898-914 (2021).</li><li>Jaminon, A. M. G., 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=Development+of+the+BioHybrid+assay:+combining+primary+human+vascular+smooth+muscle+cells+and+blood+to+measure+vascular+calcification+propensity.">Development of the BioHybrid assay: combining primary human vascular smooth muscle cells and blood to measure vascular calcification propensity.</a> Cells. 10 (8), 2097 (2021).</li><li>Reynolds, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+vascular+smooth+muscle+cells+undergo+vesicle-mediated+calcification+in+response+to+changes+in+extracellular+calcium+and+phosphate+concentrations:+a+potential+mechanism+for+accelerated+vascular+calcification+in+ESRD.">Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD.</a> Journal of the American Society of Nephrology: JASN. 15 (11), 2857-2867 (2004).</li><li>Wang, X. -. R., Zhang, J. -. J., Xu, X. -. X., Wu, Y. -. 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In this manuscript, we describe a semi-automated method for in vitro calcification determination. For this method, three critical steps of hVSMC calcification should be optimized. First, cellular density is critical for hVSMC calcification development. Low densities of hVSMCs will result in slow or no calcification and cell death due to the lack of cell-to-cell contact and the stress that is induced under calcifying conditions21. High cellular densities result in over-confluency, after wh...

Vascular calcification (VC) is an independent risk factor for cardiovascular morbidity and mortality1,2,3. Long considered a passive chemical process of ectopic mineral deposition, it now appears a modifiable tissue healing response involving the active contribution of various cells including activated vascular smooth muscle cells (hVSMC) as a driver of the disease4,5. In vivo VC can be measured by multislice CT scans as an assessment of atherosclerotic burden6,7,8. Currently, a paradigm shift is underway, wherein VC severity is becoming recognized as a risk factor in cardiovascular disease, type II diabetes, chronic kidney disease, and ageing9,10,11,12,13,14,15.
hVSMCs are the most abundant cell type in the cardiovascular system and a principal actor in the development of VC. In vitro hVSMC-induced calcification is a widely used disease model to study cardiovascular disease16,17. However, most protocols for the detection of in vitro calcification use end-point measurements that can limit data acquisition, require greater use of cellular material, and can slow research. Common methods for the detection of in vitro hVSMC calcification include the o-cresolphthalein assay, which measures solubilized calcium deposition against total protein and requires cell lysis18. Also, Alizarin Red staining is used, which binds directly to calcium deposits on fixed cells or tissue19. To study hVSMC calcification over time with either o-cresolphthalein or Alizarin Red requires batches of replicates per time point, increasing the demand on biological material, and in turn, increasing the chance of variability.
In this paper, we detail the method for the application of a novel assay that utilizes hVSMCs with a fluorescent imaging probe to determine in vitro VC progression as well as function as a singular end-stage calcification assay. We previously demonstrated that this assay is directly comparable to the o-cresolphthalein and Alizarin Red methods and can be used to distinguish between varying culture conditions20. In addition to real-time measurements, this assay may be used to determine the propensity of serum or plasma samples as a surrogate marker for clinical VC development20. This will aid in the application of biological strategies of cardiovascular sciences and disease modeling. A further application of the assay may be as a translational BioHybrid system to assess VC severity or progression from blood constituents such as serum or plasma.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcium chloride, 93%, anhydrous</td>
			<td>Thermo Fisher Scientific</td>
			<td>349615000</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6-well Clear TC-treated well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 3 System</td>
			<td>BioTek, Abcoude, The Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Merck</td>
			<td>F7524-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetuin-A-Alexa Fluor-546</td>
			<td>Prepared in-house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 Software v3.10</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Medium 199</td>
			<td>Thermo Fisher Scientific</td>
			<td>11150059</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

a semi-automated and reproducible biological-based method to quantify calcium deposition in vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

The outcome includes original images of HOECHST-stained nuclei, RFP-labeled calcification, and brightfield images. Different stages of calcification ranging from low (Figure 2) to high (Figure 3) may be detected and analyzed. Calcification can usually be spotted as black speckles using light microscopy (Figure 2D and Figure&#160;3B, arrows indicate calcification), which are useful for primary assessment...

Watch this Scientific Journal Video about A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro at JoVE.com

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

Our method allows to measure calcification propensity of patient samples using human vascular smooth muscle cells. This will help propel research into vascular calcification disorders, assisting in the development of new therapies. This technique uses a fluorescent-based detection, which increases the sensitivity and allows to measure samples over time, thereby reducing the need for replicates and increasing the robustness.To begin, culture human vascular smooth muscle cells on uncoated cell culture flasks at 37 degrees Celsius with 5%carbon dioxide. Split the cells upon reaching 70 to 90%confluency. To split, wash the HVSMCs twice with PBS.Add trypsin and incubate for 2 to 5 minutes at 37 degrees Celsius. Inhibit the trypsin reaction by adding a serum containing medium. Centrifuge the cells for 4 minutes at 350 RCF and resuspend the pellet in a maintenance medium.After preparing the cells for a split as demonstrated, count and seed the cells into a 48-well plate to start the calcification experiment. Following overnight incubation at 37 degrees Celsius with 5%carbon dioxide, gently wash the cells twice with PBS, carefully aspirating all the remaining PBS after the second wash. Gently add calcification medium and further incubate at 37 degrees Celsius with 5%carbon dioxide.Check daily with a light microscope until calcification occurs. To detect calcification via imaging, open the software and select Protocols and Create New. Select Procedure and click Set Temperature.Set the temperature to 37 degrees Celsius and the gradient to 1 degree Celsius in the pop-up window and click OK.Select the plate type of choice from the drop-down menu. Add the imaging step by clicking Image. Select the Inverted Imager and click OK.Add three imaging channels and set them to DAPI, RFP, and Bright Field.Tick the Montage box and set the desired number and location of the images. Change the Overlap to represent a better coverage of each well. Select which wells are to be imaged.A new window will open where the wells of interest can be selected. Click Focus Options to set the focus mode. A new window will open.Set each channel individually. Commonly, wells are auto-focused on the DAPI channel. Set all other channels to Fixed focal height from first channel and set by clicking OK.Close the window.Start the presetting data reduction steps by clicking Data Reduction and select Image Preprocessing. Accept the default settings by selecting OK.Set up a step to count the cells by selecting Cellular Analysis. Select TsF DAPI Images from the channel drop-down menu.Set further details after imaging has been performed. Prepare the analysis of the RFP signal by selecting Statistics in the pop-up window Label step and select TsF RFP as the input channel. Tick the Upper Value and Lower Value boxes.Tick the box for Total Area in the lower list. Select None in the Color Effect column. In the pop-up window, click Custom and tick the Background box.Press OK.For a fair readout, normalize the RFP signal per cell by pressing Ratio on the left side. In the pop-up window, select RFP Quantification Total Area. For Data Input 1 from the drop-down menu and select Cell Count as Data Input 2.Finally, select a New Data Set Name and press OK twice. Select Color Effect as demonstrated earlier, and click OK.In the upper left corner, select File and save the file as a Protocol file type. Record observations for every time point after the first instance of calcification occurs.Press Read Now in the startup software and click on Existing Protocol. Select the protocol created in the previous step. The program will prompt to save the experiment.This can be done at this stage or later manually by clicking the Save button on the upper left. A pop-up window will ask to wait for the the system to heat. Turn on the carbon dioxide gas controller and set it to 5%Once the system has reached the set temperature, a prompt will appear to insert a plate and read.Press Cancel. Transport the plate from the incubator to the imager in a safe box to avoid breakage and spillage and is in line with local biosafety regulations for transporting live cells in case of an accident. Place the plate into the plate reader.Click on Procedure. In the pop-up window, Select Preset Imaging Step. Adjust the focus and exposure on the DAPI channel first.Untick the Auto Exposure box at all channels, then click the microscope icon next to the DAPI channel. A new window will open. Select the well to direct the focus.If a signal is already visible, first, click Autofocus and then Auto Exposure. If not, first, increase the exposure and repeat Autofocus and Auto Exposure. The exposure can be adjusted manually by selecting the Exposure drop-down menu.Check the settings on multiple wells, then save the settings. Adjust the exposure in the same manner for all channels. For the RFP channel, make sure to start with a well with medium-to-high calcification.Close the procedure window and select the green Read Now button in the top task bar. Save the experiment as indicated by the software. Repeat this process for each time point.While the system reads the plate, all the images are automatically processed in the background. Adjust the settings for cell count by selecting TsF DAPI images to display. Select the well of choice by double-clicking.A new window will pop up. Select one of the images. Select the Analysis tab in the pop-up window.Select Cellular Analysis Cell Count and click OK.Deselect the RFP and Bright Field channels. Tick the Highlight Objects box. Click Options to open the menu.Adjust the Size and Intensity Threshold to include all nuclei, but exclude debris, then click OK.Click Apply Changes in the lower left to apply the settings to all images. Similarly, select a well-end image with a medium-to-high amount of calcification. To best adjust the signal-to-noise ratio, click the Analysis tab in the Task menu.Select Image Statistics. Deselect the DAPI and Bright Field channel. Click Options to open the menu.Tick the box Threshold Outliers. To set the non-subjective threshold value for the signal above the background, select the Line tool from the task bar. A window will pop up.Draw a line through a patch of signal while including an area of the background. Adjust the thresholds upper and lower values. Only count the signal above the background and exclude if there are debris or artifacts.Click OK and apply changes to transfer the settings to all other wells. Confirm that the threshold is accurately selected in the other wells. Once the cell count and RFP thresholds are set, export the data.Click the Excel button next to the drop-down menu to export the data directly into a spreadsheet. Different calcification stages ranging from low to high were detected and analyzed. Calcification was spotted as black speckles using light microscopy, which were useful for primary assessment and determining when to start imaging.For an improved signal-to-noise ratio, the processed RFP images were analyzed to quantify calcification. The data was obtained by comparing two or more conditions at the same time point. Data were normalized to cell count as calcification area per cell.The data was also depicted as a time series showing the same condition at various time points. It is important that the results after data reduction and analysis still reflect what one can see before the image transformation and thresholding.

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Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic ...