Getting Started with Plate Templates

Learn to use Plate Templates for fast analysis with minimal steps.

Plate Templates are used to analyze multiwell plate images in Empiria Studio (In-Cell Western, Absorbance Assay, and Cell Analysis). This page explains how to use the design of your multiwell plate assay to create a Plate Template in the Template Library & Experiment Designer Empiria Studio Experiment Designer and Template Library icon.

The following five points outline how a Plate Template can be used in a multiwell plate experiment.

  1. Design your experiment.

  2. Create a Plate Template based on your experimental design. Read on this page about how Plate Templates work and how to create Plate Templates.

  1. Run the experimental protocol in lab.

  2. Acquire a plate image in LI‑COR® Acquisition Software LI-COR Acquisition icon or Image Studio™ Software Image Studio icon.

  3. Analyze the plate image in Empiria Studio using the Plate Template. Read the Empiria Studio® Software Multiwell Plate Calculations Reference for more information about well quantification and calculations.

How Plate Templates Work

The plate graphic in the Experiment Designer workflow is used to select and assign wells according to their type.

  • The wells you indicate will be quantified during analysis.

  • Empiria Studio will perform calculations based on the well types you indicated (e.g., background, positive, negative, sample).

Explore: Antibody Titration Example

Click wells and Well Groups on the Plate Template image to learn more about how a Plate Template works.

Empiria Studio plate analysis template set well groups
Click the wells and Well Groups (highlighted areas) on this image to learn more about this example Plate Template. This Plate Template was designed to determine the optimal primary antibody concentration for an In‑Cell Western Assay. The experiment uses three different Well Groups (indicated by shading around the wells). Each Well Group is an identical experiment apart from the amount of primary antibody used in each group. Background wells receive no cell stain or secondary antibody. The light gray wells (those without text) contain only buffer and are called Blank wells. When this Plate Template is selected for analysis, Empiria Studio will quantify the designated wells (not blank wells).

How to Create a Plate Template

Start with a Preset Plate Template

Before creating a new Plate Template, determine if you can use a Preset Plate Template instead. Preset Plate Templates are provided with Empiria Studio in the Template Library (see Template Library & Experiment Designer for more information about the Template Library).

  • Preset In‑Cell Western Plate Templates are designed according to LI‑COR's recommended best practices.

  • Preset Absorbance Assay Plate Templates are provided for commonly used Protein Assays (i.e., assays for estimating protein concentration).

Preset Plate Templates are not editable, but you can create an editable copy by clicking the copy button next to the Preset Plate Template that you want to use.

  1. Open the Template Library Empiria Studio Experiment Designer and Template Library icon.

  2. Locate the Preset Plate Templates.

    Preset Plate Templates have [Preset] in the name and are displayed with a lock . If there are many Plate Templates, click the Status column header to sort the Template List by the status icons. This will sort all the Preset Plate Templates together in the list.

    Empiria Studio plate analysis preset templates list
    A Template Library has been sorted to group Preset Plate Templates together. Use SHIFT + Click to click a second column header to also sort by the second column.

  3. Click to open a Preset Plate Template.

    Consider your experimental design. Is there a Preset Plate Template that you could use exactly as it is?

    If there is a Preset Plate Template that is close but not exactly what you need, click copy next to the Preset Plate Template to make an editable version.

    Removing [Preset] and -Copy from the name helps keep templates organized!

Create a New Plate Template (In‑Cell Western, Absorbance Assay, and Cell Analysis)

You can also create a completely new Plate Template for an In‑Cell Western, Absorbance Assay, or Cell Analysis following the directions below. Within Empiria Studio, In‑Cell Western includes On-Cell Western Assays. The steps below contain examples based on the In‑Cell Western Antibody Titration example shown above.

Start by clicking Create Template in the Template Library, then follow the steps in the Experiment Designer workflow.

Add Details

Give the template a memorable name and helpful description for your records. Choose an Assay (In‑Cell Western, Absorbance Assay, or Cell Analysis), Well Format, and Category to help link the correct template to the image during analysis.

Select Assay first to see Category options.

  • In‑Cell Western: Use this workflow to analyze In‑Cell Western or On-Cell Western plate images. Both the In‑Cell Western and On-Cell Western are quantitative immunofluorescence assays performed in multiwell plates. Data analysis is based on whole-well fluorescence quantification.

  • Absorbance Assay: Use this workflow to analyze plate images from absorbance assays, including ELISA (enzyme-linked immunosorbent assay) and Protein Assays (i.e., assays for estimating protein concentration).

  • Cell Analysis: Cell Analysis refers to a broad range of assays for studying the function and localization of proteins in living and fixed cells. Cell Analysis covers assays that are not more precisely described as one of the other Multiwell Plate Assay types.

The Well Format is the number of wells in the plate.

Category Summary Assay
Antibody Titration Determine the antibody concentration that provides optimal signal and lowest background. In‑Cell Western, Absorbance Assay, Cell Analysis
Blocking Buffer Evaluation Determine the best blocking buffer for your experiment. In‑Cell Western, Absorbance Assay
Cell Stain Linearity Determine the range where the normalization stain is proportional to cell number. In‑Cell Western
Fixation and Permeabilization Evaluation Determine the optimal fixation and permeabilization conditions for your experiment. In‑Cell Western
Z'-Factor Determination Test the quality and robustness of an assay. In‑Cell Western
Target Analysis Determine the effect of a treatment or condition on your target. In‑Cell Western, Absorbance Assay, Cell Analysis
Protein Assay Estimate the concentration of protein in samples. Absorbance Assay

Example Plate Template 

Add Details page of the example Antibody Titration Plate Template.

This slide shows the options to select to create the example Antibody Titration Plate Template. This example shows an In‑Cell Western Plate Template. Select the correct Assay, Well Format, and Category. In particular, the Assay and Category labels help you find the Plate Template during analysis (see next slide).

 

 

Example Plate Analysis

Use details added during Plate Template creation to help find the correct Plate Template to use during analysis.
This slide shows the Example Antibody Titration Plate Template being selected during analysis. To start a Multiwell Plate analysis, navigate to your Project and click Create New Experiment. Step through the workflow to choose a Multiwell Plate > In-Cell Western > Antibody Titration Experiment. Give the Experiment a name, then move to the Select template step and choose the Example Antibody Titration Experiment.

Set Backgrounds

Designate which wells will contain background control. During analysis, background wells are quantified and automatically used to calculate background-subtracted values for other wells.

For an Absorbance Assay, background wells are required for quantification of sample wells and calculation of background-subtracted values. The Next button will be enabled once background wells have been assigned.

  1. Click and drag to select a set of background wells.

    You can also click to select individual wells.

  2. Click Set As Background.

Example Plate Template 

Background Control Well Sets in an Antibody Titration Plate Template.
This slide shows Background Control wells assigned in a Plate Template. This example shows an In‑Cell Western Plate Template.

Example Plate Analysis

Background Control Well Sets in an Antibody Titration Plate Analysis Workflow.
This slide shows Background Controls used during analysis of a multiwell plate.

Set Positive Controls

Designate which wells will contain positive controls. During analysis, positive control wells will be quantified for comparison with other wells, such as negative control wells and sample wells.

  1. Click and drag to select a set of positive control wells.

    You can also click to select individual wells.

  2. Click Set As Positive.

  3. Repeat as needed for each separate set of positive control wells.

Example Plate Template 

Positive Control Well Sets in an Antibody Titration Plate Template.
This slide shows Positive Controls assigned in a Plate Template. This example shows an In‑Cell Western Plate Template. Give Positive Control Well Sets a name that will be helpful during analysis (see next slide).

Example Plate Analysis

Positive Control Well Sets in an Antibody Titration Plate Analysis Workflow.
This slide demonstrates how information from a Plate Template is used on the Set up chart page during analysis of a multiwell plate. For example, the Series has been set to the Well Set Name, so the names "Pos" and "Neg" from the Plate Template are Series in the chart.

Set Negative Controls

Designate which wells will contain your negative controls. During analysis, negative control wells will be quantified for comparison with other wells, such as sample and positive control wells.

  1. Click and drag to select a set of negative control wells.

    You can also click to select individual wells.

  2. Click Set As Negative.

  3. Repeat as needed for each separate set of negative control wells.

Example Plate Template 

Negative Control Well Sets in an Antibody Titration Plate Template.
This slide demonstrates how Negative Controls are assigned in a Plate Template. This example shows an In‑Cell Western Plate Template. Give Negative Control Well Sets a name that will be helpful during analysis (see next slide).

Example Plate Analysis

Negative Control Well Sets in an Antibody Titration Plate Analysis Workflow.
This slide demonstrates how information from a Plate Template is used on the Set up chart page during analysis of a multiwell plate. For example, the Series has been set to the Well Set Name, so the names "Pos" and "Neg" from the Plate Template are Series in the chart.

Set Samples

Designate which wells will contain sample. During analysis, sample wells will be quantified to determine your target's response to the experimental change being studied.

  1. Click and drag to select a set of sample wells.

    You can also click to select individual wells.

  2. Click Set As Sample.

  3. Repeat as needed for each separate set of sample wells.

The Antibody Titration example does not use Sample wells.

Set Well Groups

Click and drag around a set of wells to group them for analysis. If you want all the wells on the plate to be in one group, click All Wells.

Follow these steps to create Well Groups from a sub-set of wells on the plate:

  1. Click and drag to select a group of wells.

    Select Include all backgrounds if you want to use all background wells for the Well Group you are creating. The average Total value for all background wells will be calculated and used to calculate background-subtracted values for other wells.

  2. Click Selected Wells.

    The Well Group will be created.

Example Plate Template 

Well Groups in an Antibody Titration Plate Template.
This slide demonstrates how Well Groups are assigned in a Plate Template. This example shows an In‑Cell Western Plate Template. Click and drag to select wells to group. Give the group a name that will be helpful during analysis (see next slide). The Include all backgrounds option was selected for each of the Well Groups shown here. This is indicated because the area around the background wells is not highlighted with the color used to highlight any Well Group. One background value will be calculated and used to calculate background-subtracted values for other wells.

Example Plate Analysis

Well Group information from the Antibody Titration Plate Template is used in the Experiment analysis.
This slide demonstrates how information from a Plate Template is used on the Set up chart page during analysis of a multiwell plate. For example, X-Axis labels have been set to use the Group Name from the Plate Template (see previous slide). Click Select X-Axis and Series to see information from the Plate Template that can be used as the X-Axis or series. See the In-Cell Western Analysis page for more information about the Experiment analysis workflow.