All tags Flow cyt Flow cytometry protocols for Firefly® multiplex assays

Flow cytometry protocols for Firefly® multiplex assays

​​​Find out how to set up your flow cytometer for multiplex assays with our step-by-step protocols.​


​Firefly particles should only be analyzed using the flow cytometers listed below. They require ​
green, yellow and red detectors. 

Our assays have been validated to work with the following benchtop cytometers​:

  • Millipore's Guava EasyCyte 5, 8, 12, 5HT, 6HT, 8HT, 12HT and 6-2L​​
  • BD Biosciences' Accuri C6 (ask us before use for the C6 Plus)
  • ThermoFisher's Attune® (not validated for the NxT, the multiplex immunoassay will only work with a limited range of analytes - contact us for details)

Our assays also work on most BD Biosciences' FACSCanto, LSRII and LSRFortessa™ flow cytometers, if they have the right laser / filter configuration (see below) and subject to pre-testing with our cytometer set-up kit​​.

  • All flow cytometers listed above must be properly calibrated using the Firefly particles before performing the multiplex experiment.

Millipore Guava easyCyte™ cytometers with InCyte 2.2 or later (5HT, 8HT, 11HT, 12HT, and BG-HT models)


Setup protocolsSettings files
Plate handler modelsInCyte 2.2
Tube loader modelsInCyte 2.7

InCyte 3.1

BD Biosciences' Accuri™ C6, FACSCanto™, LSRII and LSRFortessa™

Setup protocolsSettings files
FACSCanto tube loader modelsFirefly Diva 6 - pe-cy5
LSR II / LSRFortessa tube loader modelsFirefly Diva 6 - percp
C6 plate handler modelsFirefly Diva 6 - percp-cy5_5
C6 tube loader modelsFirefly Diva 7 - percp-cy5_5

C6 cytometer

Laser and filter configuration for FACSCanto™, LSRII and LSRFortessa™: either a) blue 488nm laser excitation for green Em525, yellow Em570 and red Em667 filters; or b) blue laser excitation for green and red filters, and yellow/green 561nm/532nm laser excitation for the yellow filter.


    Life Technologies Attune®

    Setup protocols

    Settings files
    Plate handler modelsAttune cytometer
    Tube loader models

    Please note that the assay is not validated for the Life Technologies Attune® NxT flow cytometer and that the multiplex immunoassay will only work with the Attune with a limited number of analytes.



    ​Don't have access to a validated flow cytometer or the necessary equipment to run our multiplex assays? You can still profile miRNAs or cytokines with the help of our sample profiling team, who have analysed over 10,000 samples.