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

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.