NeoFlow Quick Guide

1. Quick start

1. Import FCS filesClick “📥 Import FCS,” or drop FCS files onto the window.
2. Open a graphDouble-click a sample row in the left panel.
3. Set up the viewChoose the graph type, X axis, Y axis, and scales.
4. Create a gateSelect a rectangle, polygon, ellipse, or another gate tool and draw on the plot.
5. Send it to LayoutClick the “⤴” button in the Graph window.
6. Save and exportPress ⌘S, then use “Export” to create a PDF or another output.

Keyboard labels and most screenshots in this quick guide follow the macOS version. Some controls and wording differ in the Windows version.

2. FCS files and workspaces

Import FCS files

• Click “📥 Import FCS” or press ⇧⌘I. You can select multiple FCS files.
• You can also drag FCS files directly onto the Workspace window.
• If you drop a folder containing FCS files, NeoFlow searches its subfolders and imports the files into a group named after the folder.
• Open FlowJo .wsp / .jo files with “📂 Open.” NeoFlow converts groups, gates, compensation matrices, display scales, and layouts where available.

Use groups

• All Samples always contains every imported sample.
• Click “+” to create a group. Drag a sample row onto a group tab to add it.
• “Remove from Group” removes samples only from the active regular group.
• “Delete Selected” in All Samples removes the samples and their gates from the workspace.

Save the workspace

Press ⌘S or click “💾 Save” to save a .neo workspace. The file stores gates, groups, compensation matrices, axis settings, and all layouts.

3. Graphs and gates

Open a graph and change its display

1. Double-click a sample row to open a graph for the ungated population (all events).
2. Choose a graph type in the upper toolbar: Pseudocolor, Dot plot, Contour, Density, or Histogram.
3. Choose the X- and Y-axis channels, then adjust Linear, Log, or Biex scales if needed.
4. Double-click an existing gate to drill down into that population.

Create a gate

1. Select a gate tool in the lower row of the Graph window toolbar.
2. Drag or click on the plot to draw the gate.
3. For Rectangle, Polygon, Ellipse, Freehand, and Range gates, enter a name and confirm.
4. Return to the Select tool (↖) to move or reshape the gate using its body or handles.

Rename, recolor, or delete a gate

• Right-click a gate row and choose “Rename” or “Change Color.”
• Select a gate and press Delete to remove it. After confirmation, its child gates are removed as well.
• Use ⌘Z to undo and ⇧⌘Z to redo.

Apply gates to other samples

• Drag a gate row onto a row under another sample to copy the gate and its descendants.
• Drag a gate row onto a regular group tab to apply it to every sample in that group.
• Shared gates stay linked, so edits to shape, name, or color propagate to their linked copies.

4. Compensation

1. Click “🎛️ Compensation” in the Workspace toolbar.
2. Choose the target “File” and the “Population” used for inspection. The default is ungated (all events).
3. Click the off-diagonal cell you want to adjust.
4. Adjust its H or V coefficient in one of three ways:
   • Drag horizontally or vertically inside the scatter plot.
   • Move the H or V slider in the footer.
   • Enter a percentage directly in the numeric field.
5. Review the result, then save it as a preset or export it as JSON if needed.

“Auto Compensation” is on when NeoFlow starts. A saved preset is applied automatically only when the instrument information and the number and order of channels match.

5. Creating a layout

Place plots

• Click the “⤴” button in a Graph window to add the plot automatically to the current layout.
• Drag the “⤴” button onto the page to place the plot at a specific position.
• You can also drag sample or gate rows from the Workspace directly onto the Layout page.

Select, move, and resize

Refine the appearance

• Double-click a plot, or right-click it and choose “Plot Settings,” to change its population, axes, graph type, statistics, and other options.
• If multiple plots are selected, opening Plot Settings applies changes to all selected plots.
• Use “Copy Formatting” and “Paste Formatting” in the context menu to transfer display styles without changing the data source.
• Drop one Histogram onto another, or combine Pseudocolor / Dot plot tiles, to create an overlay.
• Double-click empty page space to add text. Double-click existing text or an axis title to edit it.

Create a batch report

Choose a target group, arrangement direction, and the number of columns or rows, then click “Batch.” The selected plots become templates, and NeoFlow creates a new layout for every sample in the group.

Place images, PDF, PPTX, and other files

Drop files onto the page or choose File → “Place Files in Layout...” (⇧⌘O). NeoFlow supports PDF, PDF-compatible Illustrator files, SVG, PPTX, and common raster-image formats. Copy and paste from other applications is also supported.

6. Saving, exporting, and printing

Save your editable work

Press ⌘S to save the workspace. ⇧⌘S opens “Save As.” Save the editable .neo workspace before exporting the final figure.

Export the finished output

1. Click “Export” in the first row of the Layout toolbar.
2. Select the layouts to export.
3. Choose a format: PDF, PPTX, PNG, or Illustrator (.ai).
4. Set the resolution or PPTX options if needed, then choose the destination.

Press ⌘P or click “🖨 Print” to print all layouts. Use “🖨 Print Layout” to print only the active layout.

7. Common keyboard shortcuts

8. Troubleshooting

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