A review of colour cell compensation using VenturiOne®, flow cytometry data analysis software, as endorsed by Ian Dimmick and Rebecca Stewart of the Flow Cytometry Core Facility, Institute of Human Genetics, Newcastle upon Tyne University.
Introduction to fluorochrome emmissions
Due to the increased use of flow with multiple lasers, the choice of fluorochromes available has grown considerably (1). This offers users great benefits, allowing more varied and extensive research by enabling the use of multiple lasers on individual instruments.
This offers the chance to optimise fluorochrome excitation and increase the number of fluorochromes and dyes for simultaneous detection within each experiment. The overall outcome of this being more data with fewer cells for cell compensation analysis.
When using two or more fluorochromes in an experiment, the spectral overlap of the fluorochrome emission spectra needs to be taken into consideration. Collectively the emission spectra of all fluorochromes cover a broad wavelength range (fig.1).
This will lead to spectral overlap where the fluorescence spectrum of one fluorochrome spills over into the detection channel dedicated to another fluorochrome being used simultaneously.
This spectral overlap causes difficulty when simultaneously trying to measure the true fluorescence of each fluorochrome, therefore obtaining a correct representation of the data and a correction must be applied.
This correction is termed colour compensation (1). A subtraction of the spill over from one fluorochrome onto another is applied to compensate for the spectral overlap. This value can be calculated as a percentage spillover of the primary fluorochrome into a detector in which it is not the primary detector.
Individual control samples stained with each fluorochrome to be used in the experiment are used independently to establish spectral overlap. This is in order to determine the amount of compensation required for each fluorochrome.
Researchers can adopt a method to calculate the cell compensation before data is collected by the flow cytometer called pre acquisition compensation.
Introduction to Post Acquisition Cell Compensation
Compensation can also be calculated after data collection within the software, called post acquisition compensation and if need be this can be used to check the integrity of the pre acquisition compensation values.
Post acquisition compensation means that adjustments are made away from the instrument environment at leisure, but the basics of compensation remain the same using either pre or post acquisition compensation. Using multiple fluorochromes and dyes means that compensation can be time-consuming, meaning that post acquisition is an ever increasing requirement (2).
In this review the focus is on post acquisition compensation using VenturiOne®, investigating manual and automatic post acquisition compensation methods.
In this review, I will also assess the use of compensation beads and cells which are routinely used at the flow cytometry core facility at the Institute of Human Genetics, and discusses both their advantages and disadvantages.
Pre Acquisition Cell Compensation Method with a Flow Cytometer
Please refer to Instrument User Manual for full instructions
1. Set up the instrument PMT voltages using appropriate cellular baseline controls
2. Use compensation beads to obtain maximal fluorescence of each fluorochrome whilst running under the cell derived instrument settings. The cytometer calculates compensation values. Forward Scatter (FS) and Side Scatter (SS) parameter settings may need changing, but these will not affect the final compensation values. Compensation beads are the preferred choice due to the advantage of giving distinct negative and positive peaks and increased signal intensity when analysed.
3. Verify compensation values by re analysis of the controls to ensure correct compensation values with respect to appropriate X and Y axis negative and positive mean or median values.
Post Acquisition Cell Compensation Method with VenturiOne®
Please refer to VenturiOne® User Guide for full instructions on Compensation Wizard
1. Set the default log decade scaling to match that of the pre acquisition method.
2. Open the compensation cells file into the playlist of VenturiOne®. Replicate the files for each fluorochrome that is to be compensated.
3. Open the Compensation Wizard from the compensation tab and configure the settings for compensation (See fig 2) click Next.
4. On the compensation page, adjust the gating and compensation regions to capture the populations for calculating the coefficients of the fluorochrome. See fig. 3.
5. You only need to click Next to repeat this for every fluorochrome to be compensated.
6. Then the last page of the wizard allows you to review, print or save the compensation settings file. See fig 4.
7. Save compensation file. See fig 5.
8. Repeat steps 2 to 7 with the Compensation bead files.
9. See Fig 6 for Compensation Bead matrix.
10. Manual compensation is used for minor adjustments of the compensation values.
11. Verify values with positive control using compensation matrix. See fig 7. Use V-log to check for overcompensation.
Principles of Cell Compensation for Consideration
• More accurate compensation is achieved through using compensation beads, which give brighter signals than cells.
• Autofluoresence of positive and negative populations must be the same for all compensation controls in order to achieve correct compensation (4).
• The CD19 expression expresses a weak signal. This makes it difficult to determine positive populations from negative populations. Not just a case of the brightest peak being the positive population. See fig. 8 where the uncompensated CD19 histogram shows two positive peaks. The brighter of these two is actually the spectral overlap of CD8 into the CD19 detector whereas the weaker positive peak is CD19 only.
• CD45 expression results in a very bright signal so is a good control
• Ensure to Clear Compensation after calculating each fluorochromes compensation value in VenturiOne® so that you are not calculating compensation on parameters that are already compensated. Please note, this is not required when using the Compensation Wizard.
• Compensation values must always be recalculated if the voltages of the Photomultiplier Tubes (PMT) are altered (3).
• Due to the complexity of multiple colour compensation, use the automatic compensation feature in the software primarily and use the manual feature to make minor adjustments.
• Two dimensional fluorescence plots can help determine more accurate compensation values by giving better visualization of populations requiring compensation.
• Controls for determining the compensation matrix should produce fluorescent signals that are bright as possible.
• In stem cell research, stem cells may not have well expressed surface antigens therefore they do not achieve bright fluorescent signals which we need to calculate optimum compensation values. Instead, compensation beads are recommended for calculating compensation in this scenario, as they will allow us to achieve brighter fluorescent signals.
VenturiOne® alleviates the complexity of multiple colour cell compensation, offering a quick and easy approach to post acquisition compensation, with the added flexibility of intuitive automatic and manual cell compensation options.
Try it for yourself, with a free 30-day trial here.
1. Ormerod, M (2008). Flow Cytometry- A Basic Introduction
2. Roederer, M (2001). Spectral Compensation for Flow Cytometry: Visualization Artifacts, Limitations and Caveats. Cytometry 45:194-205
3. Setting up 2 or 3 Colour FACS Analysis: http://users.ox.ac.uk/~path0116/tig/ccomp.html 18.12.97
4. Compensation (An informal perspective): http://www.drmr.com/compensation/ May 24, 2000.
With thanks to Ian Dimmick and Rebecca Stewart for dedicating the time required to collate the data for this document and for their continued support of VenturiOne®.