General Capture Assay
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:
- HoloMonitor® M4, placed in incubator
- HoloMonitor® M4 App Suite
- Culture vessel of choice with cells
- HoloLid™ for selected vessel
- Vessel holder for selected vessel
Output:
- Selected data analysis depended
Reanalysis:
- Guided assays
- In-depth analysis
PREPARATIONS
Materials:
- HoloMonitor® M4, placed inside the incubator
- HoloMonitor® App Suite software
- Cell culture vessel. Please check our list of recommended vessels.
- HoloLid™ for the selected vessel
- Vessel holder for selected vessel
- Setup and Operation Manual for HoloMonitor® M4
Steps
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.
| Vessel |
Vendor cat. number | HoloLid™ |
Final working volume |
Growth area, cm2/well |
Vessel cut in a holder |
| Sarstedt TC-dish 35 | 83.3900 | 71110 | 3.0 mL/well | 8.00 | NA |
| Sarstedt TC 6-well plate | 83.3920.005 | 71120 | 3.0 mL/well | 8.80 | top left |
| Sarstedt lumox® 24-multiwell plate | 94.6000.014 | 71130 | 1.9 mL/well | 1.90 | top left |
| Sarstedt lumox® 96-multiwell plate | 94.6000.024 | 71140 | 170 µL/well | 0.34 | top left |
| ibidi® μ-dish 35 mm, high | 81156 | 71111 | 2.5 mL/well | 3.50 | NA |
| ibidi® μ-plate 24 Well Black | 80241 | 71131 | 2.5 mL/well | 1.90 | NA |
| Eppendorf CCCadvanced® FN1 – 6 well | 0038110010 | 71150 | 3.0 mL/well | 9.40 | bottom right |
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
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
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.
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
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
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
- This section helps to reanalyze data between different assays using HoloMonitor® App Suite software.
Generating In-depth Assay results
In the Experiment overview page select the In-depth application icon for the wanted result.
Follow the respective assay protocol.
Generating Guided Assay results
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.