A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

Begin with test samples containing stimulated and fixed immune cells.

Stimulation induces the expression of specific markers in cells, whereas fixation preserves phenotypic markers, including cell surface antigens, and functional state markers, including cytokines.

Add a unique combination of heavy metal isotope-conjugated antibodies to each sample.

This technique uniquely barcodes multiple samples, distinguishing each sample by its combination of heavy metal isotopes.

The antibodies bind to a common epitope, barcoding all the cells in the samples.

Combine all the barcoded samples in a single tube, for downstream processing.

Add a metal-conjugated antibody cocktail to stain surface antigens.

Add permeabilization buffer to increase cell membrane permeability for intracellular staining.

Add another set of metal-conjugated antibodies to stain intracellular cytokines.

Barcoding enables simultaneous staining and acquisition of the pooled samples via mass cytometry. This technique maximizes assay efficiency,  improving the evaluation of phenotypic and functional state markers.

To barcode the lysed fixed cells, thaw the samples from the minus 80 degree Celsius storage, slowly on ice. Dilute the 10x barcoding perm buffer with PBS by 1 to 10, and make enough buffer for 3 milliliters per sample.

Fill one non-sterile trough with CSM and one with the 1x barcoding perm buffer. Then, add 1 milliliter of ice-cold CSM to the freshly thawed samples. Mix thoroughly, and transfer to the respective pre-labeled polypropylene cluster tubes.

Now, take a 10-microliter sample, and count the cells using an automated cell counter. Normalize the cell counts in each cluster tube by removing the volume of excess cells. Then, centrifuge the cells at 600 times g for 5 minutes at room temperature. Resuspend the cells in 1 milliliter of 1x barcoding perm buffer with a multichannel pipette, and centrifuge at 600 times g for 5 minutes at room temperature. Afterward, aspirate off the supernatant.

Line up the cluster tubes on a rack in the same order as indicated on the barcode key, so the sample matches with its barcode. Add 800 microliters of 1x barcoding perm buffer by multichannel pipette to all samples in the cluster tubes, without touching the cell pellet to reduce cell loss.

Then, set aside the rack with the cluster tubes. Remove the 20-plex Palladium barcoding kit tube strips from minus 20 degrees Celsius, and thaw at room temperature. Add 100 microliters of 1x barcoding perm buffer, mix thoroughly, and transfer 120 microliters of the resuspended barcode mix into the corresponding cell samples in the cluster tubes.

Mix thoroughly by multichannel pipette, so that there is no cross-contamination between individually barcoded samples. Incubate cluster tubes for 30 minutes at room temperature to allow the barcodes to label the cells. After 30 minutes, centrifuge the samples at 600 times g for 5 minutes at room temperature. Aspirate the supernatant. Then, resuspend them in 1 milliliter of CSM. Subsequently, centrifuge and resuspend in CSM again.

Afterward, centrifuge at 600 times g for 5 minutes at room temperature, and aspirate the supernatant. With a single pipette and using the same tip, transfer all cell pellets, and about 70 to 80 microliters of residual volume to one polystyrene tube. Do not eject the pipette tip. Set aside the single pipette with this tip.

With a multichannel pipette and new tips, add 100 microliters of CSM to each original cluster tube to maximize cell recovery. Using the single pipette with the tip that was set aside, transfer all cell pellets in 100 microliters of residual volume to the same polystyrene tube. Add CSM to top off the polystyrene tube to about 3 milliliters. Count and record the cell number of the pooled barcode set. Then, centrifuge at 600 times g for 5 minutes at room temperature, and aspirate the supernatant. Proceed to staining the barcoded samples on the same day.