LEGENDplex™ Flow Setup

Flow Cytometer Instrument Setup

In order to generate reliable data, the flow cytometer must be set up properly before data acquisition. The following sections will address machine setup for the flow cytometers listed below.

List of Flow Cytometers and Possible Configurations

Flow Cytometer	Reporter Channel	Channel Emission	Classification Channel	Channel Emission	Compensation needed?
BD FACSCalibur™ (single laser)	FL2	575 nm	FL3	670 nm	Yes
BD FACSCalibur™ (dual laser)	FL2	575 nm	FL4	660 nm	No*
BD FACSCanto™, BD FACSCanto™ II	PE	575 nm	APC	660 nm	No*
BD™ LSR, LSRII, BD LSR Fortessa™	PE	575-585 nm	APC	660 nm	No*
BD FACSAria™	PE	575 nm	APC	660 nm	No*
Cytek® Aurora with YG laser	YG1	575 nm	R4	720 nm	No^&
Cytek® Northern Lights and Cytek® Aurora without YG laser	B4	580 nm	R4	720 nm	No^&

*Compensation is not required for the specified flow cytometers when set up properly, but is recommended for consistent results.
^&Unmixing for Cytek instruments is not required.
For flow cytometers not listed here, the end-user needs to set up the machine following similar guidelines. Please refer to Setup Procedure for Other Flow Cytometers below.
The setup process typically includes the following steps. Please see the detailed setup procedure that follows, regarding your specific instrument.
1) Start up the instrument following the manufacturer’s recommendations.
2) Create a template for data acquisition using your instrument’s data acquisition software. A template is a document or worksheet with density plots that allows the user to perform machine setup and data acquisition.
3) Set up the PMT voltages of each channel to be used for data acquisition using the Setup Beads provided in the kit.
4) Determine whether compensation is needed based on the configuration of your system as shown in the table above. If compensation is needed, perform compensation using the Setup Beads provided in the kit.