Yes, that's exactly how I do it. I used to use FCAP Array but I actually find it much faster (and less frustrating) to use FlowJo and Prism. Here's how it can be done using the BD Flex kits (which have APC/APCCy7 as the bead ID, and PE as the signal - these parameters may change with other kits, but the premise is the same):

1. Acquire your CBA on FACSDiva as per manufacturer instructions (ie like a normal FACS expt).

2. Export as FCS 3.0 not FCS2 (important, since it preserves below-zero values)

3. Open in FlowJo, set a gate around the beads using FSC-SSC, then set a gate around each bead cluster using APC (x-axis) vs APCCy7 (y-axis). Use the bead product info sheets to determine which bead cluster corresponds to which cytokine (eg D5, B7 etc, with the cluster in the top right hand corner corresponding to A9)

4. Create a median fluorescence intensity statistic for PE within each of the above gates.

5. Copy and paste these gates to all of your samples and standards (perhaps flick through and check visually that they are correct for each sample).

6. Create a table of all the PE MFI stats and export it into Prism (or whatever software package you use that can perform nonlinear regression). With Prism, the line of best fit through the standards is normally perfect, and you don't need to worry about different mathematical models, just use "sigmoidal dose response variable slope" on the X=log(X) transformed data. The R-square values are typically >0.99. I've attached my CBA template file as a guide - basically paste your raw data into the Data table, and then read off your concentrations from the Results tab labelled "Read concentrations here" (the first column "X").

7. Check to make sure none of your samples are off-scale low/high. Prism has a tendency to leave empty spaces if the samples are outside the standard curve, ie it will interpolate but not extrapolate. If they are off-scale low then you can consider calling them "0 pg/ml" at your discretion. Then, adjust for your initial dilution factors (eg if you did 1:5, multiply the concentrations by 5).

Yes you can use Diva to run your CBA kit without any issues. The setup process is fairly straight forward. I believe the fluorochrome the CBA uses are APC for clustering the beads and PE for the detection and quantification of cytokine amount. First off gate SSC vs FSC to identify the beads and gate them, then do APC (Y) vs PE (X) of the bead population and you should get 6 strips of beads that cluster on the left of the x axis and move up towards the right as cytokine detection increases. Gate each strip as a cytokines and export the MFI PE fluorescence, plot out the standard curve and then use it to calculate the unknowns.

I have not used FlowJo to analyse CBA results but there is no reason that it should not work. If you expore the FCS files from you CBA kit you should be able to gate them in FlowJo as you would any other flow data.

Hope this helps and good luck.

I think the software you use to acquire your data is not that important, as long as you follow the setup steps exactly. It`s more important to have the right cytometer (they have a list of suitable cytometers in the beginning of the booklet of the kit).

You should probably be able to use FlowJo to analyze the data if you have a kit and not the flex sets, but it will take you forever. With the FCAP it take at most half an hour. If your lab orders lots of stuff from BD they might be able to give you the software cheaper. If you have the flex sets, you will probably not be able to analyze it with FlowJo, because each bead has a different size and intensity, which the FCAP software identifies. But for the kits you can figure out which bead is which from the booklet.

Yes, that's exactly how I do it. I used to use FCAP Array but I actually find it much faster (and less frustrating) to use FlowJo and Prism. Here's how it can be done using the BD Flex kits (which have APC/APCCy7 as the bead ID, and PE as the signal - these parameters may change with other kits, but the premise is the same):

1. Acquire your CBA on FACSDiva as per manufacturer instructions (ie like a normal FACS expt).

2. Export as FCS 3.0 not FCS2 (important, since it preserves below-zero values)

3. Open in FlowJo, set a gate around the beads using FSC-SSC, then set a gate around each bead cluster using APC (x-axis) vs APCCy7 (y-axis). Use the bead product info sheets to determine which bead cluster corresponds to which cytokine (eg D5, B7 etc, with the cluster in the top right hand corner corresponding to A9)

4. Create a median fluorescence intensity statistic for PE within each of the above gates.

5. Copy and paste these gates to all of your samples and standards (perhaps flick through and check visually that they are correct for each sample).

6. Create a table of all the PE MFI stats and export it into Prism (or whatever software package you use that can perform nonlinear regression). With Prism, the line of best fit through the standards is normally perfect, and you don't need to worry about different mathematical models, just use "sigmoidal dose response variable slope" on the X=log(X) transformed data. The R-square values are typically >0.99. I've attached my CBA template file as a guide - basically paste your raw data into the Data table, and then read off your concentrations from the Results tab labelled "Read concentrations here" (the first column "X").

7. Check to make sure none of your samples are off-scale low/high. Prism has a tendency to leave empty spaces if the samples are outside the standard curve, ie it will interpolate but not extrapolate. If they are off-scale low then you can consider calling them "0 pg/ml" at your discretion. Then, adjust for your initial dilution factors (eg if you did 1:5, multiply the concentrations by 5).

Thank you Adam Uldrich ! Very much appreciated! Will definitely try out for upcoming experiments.