Opteon Workflow

Press the ON/OFF button on the front panel if Opteon is not already on.

• Empty the waste tank if almost full:
  • Disconnect tubing, empty in the sink, add ~300mL bleach.
  • Reconnect waste tank to instrument.
• Refill sheath tank with DI water if nearly empty.
• Disconnect tubing, fill with DI water.
• Reconnect sheath tank to instrument.

Note: To disconnect the tubing, press the metal tab (blue arrow) and pull gently on the tubing.

Make sure Ready is displayed on the status bar.

Click File → New → New Blank Experiment.

Click File → Save As and save your file.

• We recommend saving the experiment at this time, but it is fine to save it later.
• The .ncf file should be saved in the folder assigned to your lab.

In Plate Manager, select a plate type or the tube rack.

In the Menu, click Unmix → New Reference Control Specimen.

In the new window that pops up, check Auto Update Reference Spectra, to have the unmixing recalculated whenever a gate on a reference control is modified.

Click the Edit button, next to Single Stained Controls to open the "Edit Fluorochrome" window.

• In the Edit Fluorochrome window, type the name of a fluorochrome in the box framed in green.
• Double-click the fluorochrome to add it.
  Alternatively, select the fluorochrome and click Add (framed in blue).
• Repeat for all fluorochromes used in your panel.

• Additional unstained controls are typically necessary when there are no negative events in some single color controls.
• Click the Add button (framed in red) to add an additional unstained control.
• In the image below, an "Unstained beads" control made with beads has been added.

If necessary, change the Control type and the Negative population.
As an example, in the image below, different negative populations are selected for each single-color control.

• The FITC single color control contains positive and negative events. The existing Negative population will be used for unmixing.
• For PE, the Unstained control (Type: Cells) will provide the negative population.
• For PE-Cy7, the negative population used for unmixing will be defined using the Unstained beads.

Adding the names of the markers is highly recommended!

• Obviously, the position of the controls in Experiment Manager must match the position of actual controls in the plate or tube rack.
• When using the tube rack, it is often easier to physically relocate the tubes in the rack.
• When controls are in a multi-well plate, go to Experiment manager or to Worklist and reposition the controls in the software.
• Example: The position of a sample is being edited in Experiment Manager.
• To relocate the Unstained control in well A1, go to Experiment Manager, click A1 once, then click it again.
• A1 is now highlighted in blue and can be edited (see screenshot 1).
• After typing "A3" (in place of "A1"), the Unstained sample is repositioned in A3 (see screenshot 2).
  The Plate Manager also shows the repositioning of the Unstained control.

• The A1 position is marked on the stage (top image).
• The tube rack (without any controls) sitting on the stage (bottom image).

• In Experiment Manager, double-click the first control to run.

• In the Cytometer Setting, set the Flow Rate to Slow.
• Adjust Stop condition.
• Note:After starting to run the first control, you may need to return here and adjust the Threshold.

• In Cytometer Control, click Run Single Well.
• If necessary, go to Cytometer Setting and adjust the Threshold.

• Usually, the reference controls need to be run with the same settings.
• If the settings for the first control were changed, they may need to be be applied to the remaining controls.
• In Experiment Manager, drag-and-drop the first control onto the Reference Control Specimen.
  All the settings of the first control are applied to all samples in the Reference Control Specimen:
• Cytometer setting (detector gains, flow rate, threshold)
• Fluorochrome Setting (fluorochromes, markers)
• Reference Spectra (unmixing information)
• Report (automatically generated)
• Analysys (plots, gates, gating hierarchy)
• It is also possible to drag-and-drop a sample onto another sample (not necessarily onto a specimen).

• In Cytometer Control, click Run plate.

• A window (not shown here) will pop up .
  Select the wells that contain reference controls and click Run to record them.

• Activate the Raw Workspace.
• NovoExpress automatically draws gates on Reference controls.
• On Unstained control(s) only one gate, named Main, is generated.
• Five gates are automatically generated on fluorescent controls:
  Main, Positive, Negative, Pos, and Neg.
• The Positive gate corresponds to Pos gate while the Negative gate corresponds to Neg gate.
• The Negative and Positive gates are set on the Main population.
• The Pos and Neg gates are set on the Positive population
The hierachy described above is critical for correct unmixing.
• Attention: Sometimes the Main gate does not have proper shape and cannot be scaled to define the main population of events.
• It is fine to delete the Main gate and redraw it. The newly drawn gate is automatically named Main. Note: It may be necessary to adjust the gating hierarchy!

• In Experiment Manager, look for Reference spectra under Reference Control Specimen
• Reference spectra should be shown in green font, signalling that unmixing was calculated.
• Double-click Reference Spectra
to open the Reference Spectra for Reference Control Specimen window.

• Examine the Reference Spectra for Reference Control Specimen window. Check:
• Spectral plot (all fluorochromes added to experiment should be listed!)
• Similarity Index matrix
• Spillover Spreading matrix
• If there are warnings (in red font), you may need to adjust the gates or to troubleshoot the reference controls.
• If unmixing is successful, continue with running samples

• In Experiment Manager, add specimen(s) and samples.
• In Plate Manager, click-and-drag to select wells
• Click the Tube icon in Plate Manager (framed in green) to add new samples to selected wells.
• The added samples will be immediately visible in Experiment Manager.
• In the example below, a specimen containing 5 samples was added.
• The active sample is marked with a red square in Plate manager and with a red arrow in Experiment Manager.
• Samples can be easily repositioned in Worklist or in Experiment Manager

• Click the Unmixed Workspace and add plots and gates.

Brief overview:

• Activate the first sample by double-clicking it in Experiment Manager
• In Cytometer Control, click the Run button.
• Go to Cytometer setting and make adjustments to stopping conditions or threshold, if necessary.
• Finish recording the first sample.
• In Experiment Manager, drag-and-drop the first sample onto the Specimen, to apply consistent sample properties.
• Click Run Plate, select wells and confirm.

Note: There are no screenshots added to this section. The steps described here are similar to recording single color controls.

• To export all FCS files, right-click on the Experiment Name and choose Export → Export FCS Files.
• Choose a folder to save the files and click Save.
• It is possible to export only the samples in one specimen (right-click the specimen) or a single sample (right-click the sample).

• In Experiment Manager, right-click a sample or speciment and select Export → Export Template.

• Note: Before you begin, export the Template of the relevant sample(s), specimen or whole experiment that needs to be repeated. See 6. Exporting.
• When the template is available, click File → New → New from Template.

• In the Open window that pops up, navigate to the location where the template was saved, select it and click Open.

• Select empty wells on the plate and click the New Samples on Selected Well(s) icon.

• Drag-and-drop the template settings to the new specimen.
• Rename the new samples if desired.

• You may want to run one sample first, to confirm that the the data appears as expected.
• In Cytometer Control, click Run plate

• In the pop up window (not shown), select the samples to run and confirm.

• Select 2 empty wells in the Plate Manager.
• Right-click and select Batch Creation of New Specimens.

• Navigate to the Desktop and select the Cleaning Template file on drive D:\.
• Check Import Specimen Template.
• Click OK

• Two samples will be created: Contrad and DI water.

• Place one tube with ~2 ml Contrad and one tube with ~2 ml DI water in the tube rack.
• Make sure the tubes position in the rack matches their position in Experiment Manager.

• Click Run Plate in Cytometer Control.
• In the window below, select the two cleaning wells and click Run.

• The cleaning procedure takes about 4 minutes.

• If you finish after 5 pm, check the iLab calendar to check if you are the last user of the day.

• If you are the last user of the day, press the ON/OFF button on the front panel.
  Opteon will perform a self-cleaning cycle and will power off.
• Manual shutdown can be initiated in the Instrument tab. Please DO NOT check “Clean sample injection probe” box. The Flow Core staff is responsible for cleaning the sample injection probe.