General Capture

general-capture-icon

This protocol helps to set up a General Capture Assay using HoloMonitor® M4 and the HoloMonitor®. App Suite software. The HoloMonitor® General Capture Assay provides time-lapse images for further analysis by any other HoloMonitor® App Suite assays.

Requirements:

Output:

  • Selected data analysis depended

PREPARATIONS

Materials:

Steps

calibration

Seed the cells with about 5 % confluence (ca. 6000 – 11000 cells/cm2).

►Please note that too few cells may lead to inadequate results due to auto-focus failure.

Place the vessel in the incubator and let cells attach for 2-24 hours.

Start the software and wait for complete instrument initialization.

Run an auto-calibration. With successful calibration (as shown in the picture), the instrument is ready to use.

VesselVendor cat.
number
HoloLid™Final working
volume
Growth area,
cm2/well
Vessel cut in
a holder
Sarstedt TC-dish 3583.3900711103.0 mL/well8.00NA
Sarstedt TC 6-well plate83.3920.005711203.0 mL/well8.80top left
Sarstedt lumox® 24-multiwell plate94.6000.014711301.9 mL/well1.90top left
Sarstedt lumox® 96-multiwell plate94.6000.02471140170 µL/well0.34top left
ibidi® μ-dish 35 mm, high81156711112.5 mL/well3.50NA
ibidi® μ-plate 24 Well Black80241711312.5 mL/well1.90NA
Eppendorf CCCadvanced® FN1 – 6 well0038110010711503.0 mL/well9.40bottom right
General capture

Sterilize the HoloLids™ according to the HoloLid™ sterilization and use protocol.

Add the treatment to your cells. The final working volumes per well, essential for using HoloLids™, are shown in the table above.

Slide the cell culture vessel onto the Vessel holder, its grips facing towards you. Ensure that the vessel is parallel to the holder. There is a spring that holds the vessel in place.

►When using multi-well plates, place them with the cut-off corner to the left.

Replace the standard lids with the HoloLid™.

Put the vessel holder with the sample on the HoloMonitor® M4 stage.

Select the General Capture and proceed by clicking the Setup Capture button.

EXPERIMENT SETUP

Basic setup: Describe the experiment and assign treatments to the wells

General-capture-setup

Enter the experiment name, optional experiment description and cell types.

Select the correct vessel map from the drop-down list.

Map treatments and conditions on the vessel map. Select wells by marking them with the left mouse button while moving the cursor over the relevant well/s.

Add the treatment name(s) in the text box below the vessel map and click Add/ press Enter. It is possible to add wells as individual treatments. Marked well/s are light blue, selected wells will appear dark blue.

Proceed to Capture setup.

Capture setup: Select the experiment time settings and choose capture positions

General-capture-setup-tab

Adjust the default settings for the duration, interval, and number of positions.

Ensure that the storage requirement for the experiment does not exceed the computer capacity.

When running an experiment, data needs to be stored on the computer connected to the instrument. Storing data on an external drive (e.g. connected via USB or internet server) may cause data loss due to erratic USB connections or poor internet connection.

Manual focus adjustment
MANUAL FOCUS ADJUSTMENT: The focusing tool is located in the Controls tab. Move the black square or click on the Arrow buttons to move the stage up and down. Save an adjusted focus setting for the selected position by using the Apply Current Focus button. For details, consult the Setup and Operational Manual.

Run a full or quick validation of the selected positions to ensure good image quality.

Click the positions list button to view the position list.

Inspect the images by hovering over the image icon in the list. Move the stage to that position by double-clicking the icon. If the image quality is poor, a warning sign appears. Adjust position location or focus if necessary.

When satisfied with the experiment setup, click Proceed to Capture.

Capture: Review the experiment in real-time during the time-lapse

General-capture-capture-setup

Click Start Capture.

To stop the experiment ahead of time, click the stop button.

► Note that it is NOT possible to restart the experiment once it has been stopped.

Go to the Experiments tab and open your ongoing experiment to preview the results during the run.

►Wait for the experiment to finish before starting data analysis.

When the Experiment capture finishes, click the Show Result button to get directly to the Results page.

RESULTS & ANALYSIS

Experiments tab

General-capture-results-tab

Click Experiments to see a list of the experiments.

Click on the experiment title to open an experiment summary.

Click Open to open the results page to view all images.

One experiment — multiple results

Generating In-depth Assay results

General-capture-reanalysis

In the Experiment overview page select the In-depth application icon for the wanted result.

Follow the respective assay protocol.

Generating Guided Assay results

General-capture-result
When creating a New Guided Assay result from Cell Morphology experiment – the first analysis will take some time, as the software needs to evaluate image quality and perform image analysis.

In the Experiment overview page under Guided Assay Results select Create New Result.

Choose the type of analysis in the pop-up window and name the new result.

Tick copy image analysis from and select the experiment to copy from. This will copy the image analysis settings from the selected result including all changes.

► For further data analysis steps, please see the respective assay protocol.

Press create.