The overall goal of this protocol is to quantify multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using a multiplex bead-based immunoassay and a flow cytometer. This method can be used to answer key questions in the field of immunology, such as, what are the unique cytokine profiles that orchestrate the immune response in context of specific disease states? The main advantage of this technique is the simultaneous quantification of multiple annelids can be performed using far less sample volumes than traditional ELISA formats while at the same time reducing nonspecific binding.
Demonstrating the procedure will be Nicholas Johnsen, a research associate from our product development team. Begin this procedure with biological sample preparation as described in the text protocol. To prepare the premixed antibody-immobilized beads, sonicate the premixed beads bottle for one minute in a sonicator bath at room temperature.
Then vortex for 30 seconds prior to use. If no sonicator bath is available, increase the vortex time to one minute. For standard preparation, add 250 microliters of assay buffer to reconstitute the lyophilized Mouse Th Cytokine Standard cocktail.
Mix by briefly vortexing, and allow the vial to sit at room temperature for 10 minutes. Transfer the standard cocktail to a polypropylene microcentrifuge tube labeled C7.This will be used as the top standard. Label six polypropylene microcentrifuge tubes as C1 through C6.Add 75 microliters of assay buffer to each of these tubes.
Then, transfer 25 microliters of the top standard, C7, to the C6 tube, and mix well by vortexing. This will be the C6 standard. Continue performing serial one to four dilutions, by using a new pipette tip for each tube to add 25 microliters of the previous standard to the 75 microliters of assay buffer in the next lowest standard tube.
Follow each addition by vortexing. Use assay buffer as the zero picograms per milliliter standard. To perform the assay, allow all reagents to warm to room temperature before use.
Polypropylene filter plates are used in this demonstration. It is absolutely essential the plate be kept upright during the entire assay procedure, including the washing steps, to avoid losing the beads. Pre-wet the filter paper by adding 100 microliters of 1X wash buffer to each well, and let the plates sit for one minute at room temperature.
Remove the buffer volume by using the vacuum manifold. Blot excess wash buffer from the bottom of the plate by pressing the plate on a stack of clean paper towels. Then, place the plate on top of the inverted plate cover.
For cell culture supernatant samples, add 25 microliters of assay buffer to all wells. Add 25 microliters of each standard to the standard wells. Finally, add 25 microliters of each sample to the sample wells.
Vortex the mixed beads for 30 seconds. Then, add 25 microliters of the mixed beads to each well, shaking the bead bottle intermittently to avoid bead settling. Seal the plate with a plate sealer.
Once sealed, wrap the entire plate, including the inverted plate cover, with aluminum foil. Place the plate on a plate shaker, secure it, and shake at approximately 500 RPM for two hours at room temperature. Without inverting, place the plate on the vacuum manifold and apply the vacuum as before.
Then, add 200 microliters of 1X wash buffer to each well. Remove the contents of the assay plate wells by vacuum filtration. Blot excess wash buffer from the bottom of the plate with an absorbent pad or paper towels before repeating this step one more time.
Next, add 25 microliters of detection antibodies to each well. After sealing the plate with a fresh plate sealer, wrap the entire plate, including the inverted plate cover, with aluminum foil. Place the plate on a plate shaker and shake at approximately 500 RPM for one hour at room temperature.
Without vacuuming, add 25 microliters of streptavidin phycoerythrin reagent directly to each well. Seal and wrap the plate as before. Then, place the plate on a plate shaker and shake at approximately 500 RPM for 30 minutes at room temperature.
After repeating the vacuum filtration step twice, add 200 microliters of 1X wash buffer to each well. Re-suspend the beads on a plate shaker for one minute. Using a multichannel pipette, transfer the samples from the filter plate to FACS tubes in order to read the samples on a flow cytometer.
Start up the flow cytometer and acquisition software according to the manufacturer's instructions provided with the instrument. Create a dot plot with forward scatter on the X-axis and side scatter for the Y-axis. Set forward scatter and side scatter to linear mode.
Vortex the vial of raw beads included in the kit for 30 seconds to re-suspend the beads. Transfer 400 microliters of the raw beads to a new FACS tube. Set the flow cytometer flow rate to low.
Run the raw beads, carefully adjusting the gain and voltage for forward scatter and side scatter so that both size populations of these beads are visibly separated and easy to gate. Adjust the forward scatter threshold to exclude unwanted events. In the forward scatter versus side scatter plot, draw a gate that includes all bead populations.
Display the gated bead populations from the forward scatter versus side scatter plot on the second dot plot with PE on the X-axis and APC for the Y-axis. Adjust the PMT voltage for the APC fluorescence channel so that the APC signal for all bead populations has a median fluorescence intensity, or MFI, that lies between 10 and 5, 000. Next, vortex the vial of PE setup beads for 30 seconds to re-suspend the beads.
Transfer 400 microliters of the re-suspended PE beads to a new FACS tube. Replace the raw beads tube in the flow cytometer with the PE beads tube. Adjust the photomultiplier tube voltage for the PE fluorescence channel setting so that the MFI of the PE beads falls between the lot-specific range found listed on the PE beads vial.
To perform data acquisition, set the number of bead events to be acquired to about 300 per anolyte. Vortex each sample for five seconds before analysis. Then, read the samples.
When reading the samples, set the flow cytometer to setup mode first, and wait until the bead population is stabilized before switching to acquisition mode. Export only the gated events rather than total events before performing data analysis as described in the text protocol. Representative standard curves for target anolytes were generated using the mouse T helper cytokine panel.
Properly run assays will demonstrate curves with broad dynamic ranges as shown here. Standard curves must be run each time the assay is performed. Mouse splenocytes were cultured under various conditions for 48 hours.
Culture supernatants were collected, and then concentrations of 13 targets were quantified using the mouse T helper cytokine panel. Differential cytokine expression profiles, in response to stimulation conditions, are clearly visible. Once mastered, the laboratory benchwork of this technique can be completed in less than five-and-a-half hours if performed properly.
The data acquisition and downstream analysis times will vary depending on the number of samples. After watching this video, you should have a good understanding of how to employ this protocol, quantify anolyte concentrations in biological samples using a cytometric, bead-based immunoassay. This includes reagent preparation, incubation steps, and flow cytometer setup for data acquisition.
