Whether you want to multiplex up to 500 different markers in one reaction or develop lower plex assays, knowing how to make a multiplex bead mix from different types of bead stocks is critical for proper assay performance.
While non-conjugated Luminex® beads can be purchased as individual bead stocks at different bead concentrations, after being coupled with different capture molecules for genomic or proteomic applications, Luminex protocols recommend resuspending coupled beads to 50 million beads/ml or less (50 million/ml = 50,000 beads/μL; see xMAP® Cookbook coupling protocols). If using MagPlex®-TAG™ beads for certain genomic assays, they are available at 2.5 million beads/ml. Whether coupling your own beads or purchasing MagPlex-TAG beads, it is recommended that the multiplex mix delivers about 2,500 beads of each region per reaction—however, this amount can be increased or decreased as needed depending on the sensitivity of an assay’s chemistry.
Using multiple individual bead stocks to make multiplex master mixes delivering the correct number of beads per reaction can be tricky and prone to calculation errors, since each chemistry and experiment design requires unique numbers of bead regions per reaction (plex) with specific volumes of a bead mix per reaction to give a specific number of beads/region in each reaction. To help new and experienced users accurately make multiplex bead mixes for assays with different requirements, an Excel-based bead calculator is available for determining how to make multiplex bead mixes from individual bead stocks or concentrated multiplex bead mixes.
The Excel file contains four tabs. Two of the tabs are for determining how to make a ready-to-use multiplex bead mix from multiple individual bead stocks. The from individual bead stocks tab is without a % overage correction (Figure 1A) while the individual bead stocks+X% tab factors in a user defined % overage correction for pipetting errors (Figure 1B). The two remaining tabs are for calculations to make multiplex bead mixes from concentrated multiplex bead stocks that already contain multiple bead regions. These are the from multiplex bead stock tab (Figure 2A) and the from multiplex bead stock+X% tab for calculations with a user defined % overage to compensate for pipetting dead volume (Figure 2B).
Figure 1. Tabs for using individual bead stocks with and without adding a % overage to compensate compensate for pipetting errors.
All four of these tabs have a standard layout for required data entry by the user in the green cells, with instructions for making the mixes and the volumes needed indicated in the blue fields. After information about certain assay parameters are entered in the green cells, the volume information in the blue fields is updated automatically to indicate if concentration is needed or not. This directs the user to the proper protocol with the required instructions and volumes needed to make the ready-to-use bead mix.
Figure 2. Tabs for using concentrated multiplex bead stocks with and without adding a % overage to compensate for pipetting errors.
Most of the assay parameter information the user provides is similar, whether starting with multiplex stocks containing multiple bead regions (Figure 3A) or using individual bead stocks for every bead region (Figure 3B). The one difference is when using multiple individual bead stocks you need to enter the number of bead regions that will be in the final multiplex mix as “# of bead regions needed =” (Figure 3B). All other data the user enters is similar for both starting bead stock types. In addition, the +X% tabs have an extra field for entry of a user defined % overage to allow minimum increases in the total mix volume to compensate for pipetting dead volume.
Figure 3. Layout of user required assay parameters to enter for making bead mixes from multiplex bead stocks or individual bead stocks.
For assistance with any of the spreadsheet’s functions and data entry, there are mouse over pop up instructions for several cells on all of the tabs as shown in Figure 4. Any cell that has a red mark in the upper right corner has useful pop up information or instructions. It is recommended that you review the pop up instructions on the tabs for the type of stocks you will be working with before using any of the tabs. You can also contact your Field Application Scientist or Technical Support for assistance.
Figure 4. Example of mouse over pop up instructions.
Once you are familiar with the layout and entering the required assay parameters, getting instructions on how to make a ready-to-use custom multiplex mix is easy.
For example, the user is designing a 32-plex assay and has conjugated stocks for 32 individual bead regions. For most of the bead regions you have 1.5 ml of each with a bead concentration for each region of 2.0×106 beads/ml (note that all the individual bead stocks must have the same bead concentration/ml, even if it is more or less than the 2.0×106 beads/ml used in this example). However, from previous assay optimization studies, 500 ul was used from a few of the bead stocks, leaving 1 ml total volume for some of these regions. This will require the user to enter 1,000 in the “total volume (in ul) of each region’s stock =” field on any of the tabs. The assay also requires 10 ul of the multiplex bead mix/reaction to deliver 2,500 beads/region for each reaction and enough mix is needed for 600 reactions.
For this type of assay, one of the individual bead stocks tabs can be used. If using the from individual bead stocks tab, the assay parameters outlined above are entered as shown in Figure 5, where the results direct the user to make the mix by concentrating and shows the required steps and volumes (Figure 5). Also note that to make enough mix for 600 reactions there is enough of all of the individual stocks, as indicated by the yes in cell C10 (Figure 5). If the mix is needed for 900 reactions, cell C10 would say no, indicating there is not enough of all the bead stocks to make a mix for 900 reactions. In situations where there is not enough to make the multiplex bead mix, the user can make more of the specific bead conjugates if that is what is being used in the assay, or purchase more MagPlex-TAG beads if these are being used for a genomic assay. Another option is to decrease the “number of reactions desired.”
Figure 5. Example of using the spreadsheet to make a 32-plex mix with 2,500 beads/region/reaction for 600 reactions.
If using a concentrated multiplex bead stock containing all the bead regions needed in the stock, similar assay parameter data would be entered into one of the from multiplex bead stock tabs to determine how to make a ready-to-use multiplex mix and what volumes are needed. On the from multiplex bead stock tabs there is no need to specify the number of bead regions in the multiplex stock, however, all of the bead regions must be at the same number of beads/ml in the multiplex stock.
When making a multiplex stock mix for large numbers of reactions or automated applications, it may be handy to make more bead mix than actually needed to prevent running short when dispensing the mix. This can be done by selecting more reactions in the “number of reactions desired =” fields or you can use one of the tabs where the user can enter a % overage. If using one of the +X% tabs, just enter the desired % overage as a whole number to allow for more mix to be made to compensate for pipetting errors. For example, if you want to make 5% more of the bead mix than is needed for the desired number of reactions, just enter 5 in the field for “% overage desired =.” Using these +X% tabs allows the user to adjust the amount of overage more sparingly to minimize wasting beads.
Once you have reviewed the pop up instructions and know how to enter data about the assay’s design parameters, you are ready to quickly and accurately determine how to make a suitable, ready to use multiplex bead mix. If you need additional assistance with using this tool, contact your Field Application Scientist or Technical Support.
To find out how you can develop your own multiplex assays, click here to instantly download the 2nd edition of the xMAP Cookbook.