FAQs

The biological samples I used in my LEGENDplex™ assay are BSL 2+. Can I fix them prior to flow cytometer acquisition in order to sterilize my samples?

The biological samples themselves cannot be fixed before the assay. However, we have found that fixing the beads after the assay with 1-4% paraformaldehyde (PFA) in 1X PBS prior to reading the beads on a flow cytometer is compatible with the assay. Incubation time with PFA should not exceed 10-15 minutes. Prolonged incubation of beads with 1-4% paraformaldehyde will decrease the signal dramatically. Immediately following fixation, the PFA should be washed out using the 1X Wash Buffer supplied with the kit. Beads should be resuspended in the 1X Wash Buffer supplied with the kit.

Does LEGENDplex™ software run on Macs and PCs?

Yes. Our software is compatible with both Macs and PCs. Get more details and download the software at: www.biolegend.com/legendplex/software.

My standard curve is not linear; can I use the non-linear part of the standard curve during analysis?

Yes. It is possible to use the non-linear part of the standard curve for calculation of results. The LEGENDplex™ data analysis software uses a five-parameter curve-fitting algorithm, which determines the minimum and maximum detection concentrations of each target and reports them. If sample concentrations fall outside of the maximum detectable range, the sample will have to be diluted and re-analyzed.

Is special software required for data analysis?

Typically flow cytometers generate output files in FCS format (e.g. FCS 2.0, 3.0, or 3.1) and in some cases in list mode file format (LMD). Other software may be available to analyze FCS files. Data generated using LEGENDplex™ kits can be analyzed using the freely available LEGENDplex™ data analysis software. Please check our website for the most updated versions of the software.

What is the shelf life of LEGENDplex™ kits?

LEGENDplex™ kits are guaranteed for 6 months from the date of receipt, but may have a shelf life of up to 2 years from the date of manufacture.

What curve-fitting algorithm can I use for generating my standard curve?

The LEGENDplex™ data analysis software uses a default five-parameter logistic curve-fitting algorithm, which determines sample concentrations and calculates minimum and maximum detection concentrations of the standard curve. If the default algorithm does not work for a particular set of data, other curve fitting methods are available in the software options. If sample concentrations fall outside of the maximum detectable range, the sample will have to be diluted and re-analyzed.

What is the difference between LEGENDplex™ and Luminex® Assays?

LEGENDplex™ and Luminex® are both bead-based multiplex immunoassays that use the basic principles of sandwich immunoassays. Both systems use fluorescence-coded beads to achieve multiplexing. The major difference is in the data acquisition. LEGENDplex™ uses common lab flow cytometers for data acquisition, whereas Luminex®-based assays have dedicated machines for this purpose. Therefore, one of the advantages is that LEGENDplex™ can be run on widely available flow cytometers without needing specialized machines. As such, LEGENDplex™ cannot be run on Luminex® machines.

When do I need to do instrument compensation?

If a single laser instrument is used for both reporter (PE) and classification (PE/Cyanine5), then compensation is needed to resolve the signal spillover from PE to PE/Cyanine5 and vice-versa. Even on dual laser instruments, some compensation may be required. Please check your machine for the need for compensation. When compensation is required, use the provided calibration beads and follow the Flow Cytometer Setup instructions in the LEGENDplex™ manual. In addition, adjust the PMT voltages such that there is good bead separation, the bead populations are not out of scale, and the PE signal is appropriately amplified to ensure proper signal sensitivity.

The kit instruction manual says that the kit is validated for serum, plasma, and culture supernatant sample types. Can I also use tissue homogenates with LEGENDplex™?

Yes, all of the above sample types may be used with LEGENDplex™ kits as long as they are prepared in a manner compatible with immunoassays. For tissue homogenate samples, they should be prepared in a neutral pH buffer containing no denaturing chemicals (e.g. SDS, urea, thiourea, cholate, etc.) and no ionic detergents and minimal amounts of non-ionic detergents (e.g. NP-40) may be used in sample preparation. The tissue/cell homogenization buffer should not contain excessive ionic strength above physiological concentrations of salts. If possible, the buffer should also contain protease inhibitors. In addition, the samples should be centrifuged to remove particulates.

Should I perform the assay with the filter plates or with V-bottom plates?

Filter plates or V-bottom plates have been included in some kits for your convenience. A vacuum filtration unit is required to work with the filter plates. However, if you don’t have access to a vacuum manifold or if you prefer, then you can use the V-bottom plates and follow the recommended assay protocols for the type of plates you choose. All plates should be made from low binding polypropylene. Polystyrene ELISA or cell culture plates should not be used.

What are the compatible flow cytometers for the LEGENDplex™ assays?

LEGENDplex™ assays can be used on most commonly used flow cytometers that can read APC and PE, such as BD FACSCalibur™, BD FACSCanto™, BD FACSCanto™ II, BD LSR I™, BD LSR II™, BD LSRFortessa™, BD FACSAria™ I, II, III, BD Accuri C6™, BD FACSVerse™, Gallios™, CytoFLEX™, Moflo XDP™, Attune™NxT, NovoCyte™, MACSQuant®, and Guava® easyCyte. Please refer to the “Materials to be provided by end-user” section of your LEGENDplex™ manual for details on the requirements of the machines in terms of laser and channel configurations. For other brands of flow cytometers, the end-user needs to make sure that the machine is set up properly before use.

If I don't have a vacuum, how do I remove the liquid from my plate?

If you do not have a vacuum, the assay should be run in a V-bottom plate. After centrifugation using a swinging-bucket rotor with a plate adaptor, you can remove the liquid by flicking the plate quickly, dumping the contents into a sink, and patting it dry carefully on a stack of clean paper towels without losing the beads. Alternatively, you can remove the liquid by using a pipette.