In‑Cell Western™ Assay Analysis

Image display options are available above the image. Only the image display is changed; the original image file remains the same.

• Zoom: Click the zoom buttons above the image to make the displayed image smaller or larger . A zoom level, shown as a percentage relative to the size of the software window, is shown next to the zoom buttons.

• Display Settings: Click to change display settings.

• Display Channels: Click to select the channels you want to display.

• Display Well IDs: Click to stop showing Well IDs.

• The plate grid is the grid of well circles inside a rectangular outline overlaying the image. The plate grid is used to specify well size and location.

• Only well circles that correspond to the wells designated in the Plate Template are quantified.

• Well circles that correspond to wells designated in the Plate Template are identified (by default) with a Well ID label.

• Ensure the well circles being quantified are placed on top of the wells on the image that need to be quantified.

• Adjust well size: Click Decrease or Increase to change the size of all the well circles on the image.

• Adjust plate grid rotation: Click to rotate the entire plate grid counter-clockwise or to rotate the entire plate grid clockwise.

• Adjust plate grid location: Click the rectangular outline of the plate grid. Drag the plate grid so that the well circles in the plate grid cover the appropriate wells on the plate image.

• Choose the channel to use for normalization from the Normalization Channel list.

  For example, this will be the channel with a cell stain such as CellTag™ 520 Stain or CellTag 700 Stain.

• The Signal values from this channel will be used to calculate normalized values in other channels.

  See Signal Analysis Calculations for more information about normalization calculations.

You may not want to quantify all the wells that were designated for quantification in the Plate Template.

• If there is a well you do not want to quantify, select the well and then click Exclude Well.

• To quantify a well that had previously been excluded, select the well and then click Include Well.

If you select Use trim value calculations, the pixels with the highest and lowest 5% of pixel intensity values in each well will be excluded from the calculation of Total and Signal. Formulas that normally use Total and Signal will instead use Trim Total and Trim Signal.

It is up to the researcher to decide if Trim values should be used. See Signal Analysis Calculations for more information.

Click Analysis Table to view well quantification data for assigned wells, including Total, Signal, Background, and Normalized Signal.

Click to export all the data to a spreadsheet.

See Signal Analysis Calculations for more information about the quantification data in the table.

1. Click Start New.

2. In the Start New Chart dialog:

   • Select Normalized.

   • Enter a unique Chart Name that will help you identify this chart in a list of charts.

   • Select the Channel that contains the target to be analyzed.

   • If a chart is already displayed, you can select Use current chart settings to use the X-Axis, Series, and text from the current chart on the new chart.

3. Click Start.

4. Fill in the appropriate X-Axis and Series information in the data table.

   This can be done manually, or by clicking Select X-Axis and Series to select information from the Plate Template.

   In the Select X-Axis and Series dialog:

   1. Select from the X-Axis options, then click Apply.

   2. Select from the Series options, and then click Apply.

   3. Click Close.

5. Other chart modifications can be made as needed.

   For example, a Chart Title can be added and you can choose where to place the chart key.

These instructions apply to Fold Change or Percent Change. The term "Fold Change" is used for short, instead of saying "Fold Change or Percent Change".

The scatter plot options in Multiwell Plate Experiment workflows do not use Trim Signal values. Trying to use one of those scatter plot options with a well that has negative signal values can cause problems with the chart display.

1. Click Start New.

2. In the Start New Chart dialog:

   • Select Fold Change.

     You will have the option to choose how the fold change is calculated later.

   • Enter a unique Chart Name that will help you identify this chart in a list of charts on the Setup chart page.

   • Select the Channel that contains the target to be analyzed.

   • If a chart is already displayed, you can select Use current chart settings to use the X-Axis, Series, and text from the current chart on the new chart.

3. Click Start.

4. Choose how to calculate Fold Change. In the table above the chart, select Control for the Well Set that you want to use as the control value in the Fold Change calculations. See Signal Analysis Calculations for more information.

   

5. Fill in the appropriate X-Axis and Series information in the data table.

   This can be done manually, or by clicking Select X-Axis and Series to select information from the Plate Template.

   

   In the Select X-Axis and Series dialog:

   1. Select from the X-Axis options, then click Apply.

   2. Select from the Series options and, then click Apply.

   3. Click Close.

6. Other chart modifications can be made as needed.

   For example, a Chart Title can be added and you can choose where to place the chart key.

Some data cannot be included in a chart

You may see the following message at the top of the Set Up Charts page.

Some data cannot be included in a chart. Click this link for more details.

This message indicates that data from at least one Well Group cannot be included in a chart. The check box in the Include column will be unavailable for Well Groups that cannot be included in a chart.

Possible solutions

1. Ensure that at least two background wells are designated in the Plate Template for the problematic Well Group.

2. Ensure that the background wells are being quantified on the Quantify wells page. Wells that have been excluded from quantification are marked with this symbol: .

3. If something is wrong with the wells and you do not want to include them in your analysis, it may be best to run the experiment again.

Charts cannot be created

You may see a banner with the following heading on the Set Up Charts page.

Chart cannot be created.

If you see this message, start with the following steps to identify and resolve the problem.

Possible solutions

1. On the Quantify wells page, ensure that a normalization channel is chosen in the Normalization Channel list.

2. In the Plate Template, ensure that background wells and at least one other type of well (positive, negative, sample) have been designated.

3. On the Quantify wells page, ensure that the background wells and the wells of the other well type are being quantified. Wells are not quantified if they have been "excluded". Wells that have been excluded are marked with this symbol: . If a well that you want to include has been excluded, select the well and click Include.

4. If something is wrong with the wells and you do not want to include them in your analysis, it may be best to run the experiment again.

During the development of an In‑Cell Western Assay, it is important to determine the relationship between cell number and signal intensity to ensure that signals are detected within the linear range of the assay.

To determine the cell stain linearity for an In-Cell Western Assay, first design a Cell Stain Linearity experiment, and set up a Plate Template (with the Cell Stain Linearity category) in the Empiria Studio Experiment Designer.

See the [Preset] Cell Stain Linearity Plate Template in the Template Library for an example experimental design. You may find it helpful to create an editable copy of the [Preset] Cell Stain Linearity Plate Template for your experiment. Create an editable copy by clicking the copy button next to the [Preset] Cell Stain Linearity Plate Template in the Template Library.

On the Find linear range page, a graph shows the relationship between cell number and signal intensity. Click a channel in the Channel list to view the graph for that channel.

A colored bar above the graph indicates whether the relationship between cell number and signal intensity has excellent linearity, good linearity, or no linearity.

• Green sections on the bar correspond to regions on the graph where there is excellent linearity between cell number and signal intensity.

• Yellow sections on the bar correspond to regions on the graph where there is good linearity.

• Red sections on the bar correspond to regions on the graph where the relationship between cell number and signal intensity is not linear.