Cytometric Bead Array (CBA) Mouse Th1/Th2 Cytokine Kit From BD

A standard method for measuring soluble cytokine production from cells is to use an ELISA specific for the cytokine of interest. These can be timely and involve making sample dilutions to effectively quantify the cytokine amount. Also, conventional ELISA plates typically measure only one cytokine at a time for a given sample. BD’s Cytometric Bead Array (CBA) is designed to not only avoid extensive sample dilutions and timely protocols, but also to measure five different soluble cytokines at once. The five cytokines: IL-2, IL-4, IL-5, IFN-gamma, and TNF-alpha, represent cytokines commonly associated with Th1 and Th2-type immune responses, and therefore can be used to assess this polarization in a given sample.

The kit contains five bead types, each coated with a specific antibody for one of the five cytokines, and each with a distinct fluorescent intensity. All five beads are incubated with each sample or standard (serial dilutions made from stock provided in kit) along with PE (Phycoerythrin)-conjugated detection antibodies. After incubation and a wash step, samples are run on a flow cytometer. The kit provides reagents to run two standard curves and 50 test samples.

I have used this kit to detect changes in cytokine production from murine T cells after various pharmacologic treatments. Typically, I culture the cells for 48 hours, but have also used a 24 hour timepoint, and both of these result in cytokine levels within the limits of detection. Primarily, I use anti-CD3 as the T cell stimulus, and this induces a Th1 response, while changes to the Th2 cytokines are undetectable (levels too low). However, after PMA stimulation, the Th2 profile is within the limits of detection.

I have used this kit nearly 100 times (not an exaggeration!), and while I have obtained great results, it hasn’t come trouble-free. First of all, a few (or more) dilution tests should be performed to determine where your sample levels fall within the standard curve. Also, I recommend running samples at least in duplicate and averaging the results, as I have found variation between within duplicates or triplicates. Under certain treatments, my samples produce very low amounts of some of the cytokines, and therefore it is better to extend the standard curve three more dilution points below the lowest recommended standard.

Also, the so-called “required” (and very expensive) BD CBA Software is not always consistent, in that a sample with a PE-Mean value significantly lower than another sample will actually come out of the software analysis with a pg/ml value significantly higher than the other sample. There is clearly something wrong with this analysis, and I have found it is much more reliable to do all the analysis in an Excel spreadsheet instead (plugging PE-mean values into the linear equation of the standard curve). There have also been several experiments where my results are completely off (excessively high or excessively low pg/ml values) from previous experiments using samples of the same kind. I don’t think this is an attribute of the kit itself, but nonetheless, I warn you that you might have to use a couple (or more) kits to optimize your sample preparation and obtain results free of wacky outliers.

Overall, the kit can provide great results on five different cytokines at once in about 6 hours (time it takes to prepare about 50 sample dilutions, set up tubes with beads and PE-antibody, incubate, wash and run). The kit is expensive, but because it multiplexes, I think it is comparable to, or perhaps even less expensive than the cost of 5 different ELISAs. The kit does require the use of a flow cytometer, and software templates need to be set up on this machine for running the samples. I run my samples on a BD LSR II, and my BD sales rep was very helpful with settting up the software. Once optimization is established, it is a good method to use to detect Th1/Th2 cytokine production.