BD FACSDiva: Standard Operation Protocol SOP... · 2013-01-23 · LKS Faculty of Medicine Version 1.1 University of Hong Kong BD FACSDiva Operation Manual – Spectral Overlap & Compensation
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LKS Faculty of Medicine Version 1.1
University of Hong Kong
BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO
1
BD FACSDiva: Standard Operation Protocol
Spectral Overlap & Compensation – PROGRAMME-AIDED
User should understand the Basic Operation before proceeding to the following steps.
For the demonstration, we will assume this is a 4-color flow cytometric analysis
experiment. These fluorochromes include FITC, PE, PerCP, and APC.
1. Determine the fluorochromes used in the experiment.
Select Experiment > Compensation Setup > Create Compensation Controls
LKS Faculty of Medicine Version 1.1
University of Hong Kong
BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO
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2. Highlight to ‘Include separate unstained control tube/well’. Click ‘OK’ to continue.
3. Click on “+” to expand the ‘Compensation Controls’
LKS Faculty of Medicine Version 1.1
University of Hong Kong
BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO
3
4. Select ‘Unstained Control’ and press ‘Load’ (or ‘Run Single Tube’ in FACSCantoII).
Adjust the gate position and PMT voltage for each fluorochrome. Their signal peaks
should stand BELOW 102 on their corresponding channel scale. Press ‘Record Data’ to
store sufficient number of cells for further analysis.
LKS Faculty of Medicine Version 1.1
University of Hong Kong
BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO
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5. Select ‘APC Stained Control’ and press ‘Load’. DO NOT ADJUST THE PMT
VOLTAGE. Press ‘Record Data’ to store sufficient number of cells for further analysis.
6. Select ‘PE Stained Control’ and press ‘Load’. Adjust the gate position, if necessary.
DO NOT ADJUST THE PMT VOLTAGE. Press ‘Record Data’ to store sufficient
number of cells for further analysis.
LKS Faculty of Medicine Version 1.1
University of Hong Kong
BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO
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7. Repeat Step 6 for the other Single Stained Controls. Again, adjust the gate position
only if necessary. DO NOT ADJUST THE PMT VOLTAGE. Press ‘Record Data’ to
store sufficient number of cells for further analysis.
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LKS Faculty of Medicine Version 1.1
University of Hong Kong
BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO
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8. Go to Experiment > Compensation Setup > Calculate Compensation
9. Click ‘Apply Only’ to set the Specimen within Experiment to use this compensation
model derived from these single stained tubes.
10. Click ‘New Specimen’ to create tubes for a new set of samples.
11. These samples should have a compensation matrix finely set at its optimal level, with
their fluorochromes properly compensated.
LKS Faculty of Medicine Version 1.1
University of Hong Kong
BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO
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12. A mixture of fluorochromes was run to demonstrate the significance of compensation.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
COMPENSATED Sample
UNCOMPENSATED Sample
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