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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
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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

Feb 20, 2020

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Page 1: 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

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

Page 2: 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

LKS Faculty of Medicine Version 1.1

University of Hong Kong

BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO

2

2. Highlight to ‘Include separate unstained control tube/well’. Click ‘OK’ to continue.

3. Click on “+” to expand the ‘Compensation Controls’

Page 3: 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

LKS Faculty of Medicine Version 1.1

University of Hong Kong

BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO

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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.

Page 4: 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

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.

Page 5: 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

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.

________________________________________________________________________

Page 6: 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

LKS Faculty of Medicine Version 1.1

University of Hong Kong

BD FACSDiva Operation Manual – Spectral Overlap & Compensation -AUTO

6

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.

Page 7: 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

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