A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

1. Barcode of Lysed/Fixed Blood Cells

1. Thaw the lysed/fixed cell samples from the -80 °C storage slowly on ice; a maximum of 20 samples can be labeled with unique barcodes and pooled using this system. Dilute the 10X barcoding perm buffer by 1:10 with PBS; make enough buffer for ~3 mL per sample.
2. Fill one non-sterile trough with the CSM and one with the 1X barcoding perm buffer. Add 1 mL of ice-cold CSM to freshly thawed samples, mix thoroughly with a pipette, and transfer to the respective pre-labeled polypropylene cluster tubes.
3. Take a 10 μL sample to count cells using an automated cell counter or hemocytometer. Normalize the cell counts to 1.5–2 x 10⁶ cells per sample in each cluster tube: remove and discard the volume of excess cells above 2 x 10⁶ cells.
4. Centrifuge the cells at 600 x g for 5 min at room temperature. Resuspend the cells in 1 mL of 1X barcoding perm buffer with a multichannel pipette and centrifuge at 600 x g for 5 min at room temperature. Aspirate off the supernatant.
5. 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 μL 1X barcoding perm buffer by multichannel pipette to all samples in the cluster tubes without touching the cell pellet with the pipette tips (no mixing) to reduce cell loss. Set aside the rack with the cluster tubes.
6. Remove 20-plex Pd Barcoding Kit tube strips from -20 °C and thaw at room temperature. Add 100 μL of 1X barcoding perm buffer, mix thoroughly, and transfer 120 μL of the resuspended barcode mix into the corresponding cell samples in the cluster tubes.
7. Mix thoroughly by multichannel pipette so that there is no cross-contamination between individually barcoded samples. Incubate cluster tubes for 30 min at room temperature to allow the barcodes to label the cells.
8. Centrifuge at 600 x g for 5 min at room temperature. Aspirate the supernatant, then resuspend in 1 mL of CSM.
9. Centrifuge and resuspend in CSM again.
   Note: Each sample is now labeled with a unique barcode, and samples are ready to be pooled.
10. Centrifuge at 600 x g for 5 min at room temperature and aspirate the supernatant. With a single pipette and using the same tip, transfer all cell pellets in ~70–80 μL residual volume to one polystyrene tube. Do NOT eject the pipette tip; set aside the single pipette with this tip.
11. With a multichannel pipette and new tips, add ~100 μL CSM to each original cluster tube to maximize cell recovery. With the single pipette with the tip that was set aside, transfer all cell pellets in ~100 μL residual volume to the same polystyrene tube.
12. Add CSM to top off the polystyrene tube (~3 mL). Count and record the cell number of the pooled barcode set. Centrifuge at 600 x g for 5 min at room temperature and aspirate the supernatant. Proceed to stain the barcoded samples on the same day.
    Note: It is normal to expect ~20–30% cell loss in the barcoding process.

2. Staining of Barcoded Lysed/Fixed Blood Cells and Preparation for Analysis on Mass Cytometry Instrument

Note: Each 1X titer of staining antibody (1 μL of antibody per 100 μL staining reaction), can usually stain 3–4 x 10⁶ cells. Therefore, when all barcoded samples are pooled into one tube, the amount of antibody must be scaled up. If 20 barcoded samples amount to 30 x 10⁶ cells, and each 1X titer can stain 3–4 x 10⁶ cells, the barcoded sample only requires a 10X titer, as opposed to staining each sample individually, which would require a 20X amount of antibody (1X per individual tube). The concentration of the antibody to cell number should be carefully titrated for each individual antibody cocktail (not discussed here).

1. Stain the cells using antibodies (Table of Materials) for surface staining and cytokine induction.
   1. Make the surface staining cocktail by calculating the amount to be added and accounting for pipetting error (i.e., if staining 20 barcoded samples of 2 x 10⁶ cells in each sample, a 10X titer stain is required; for final volume calculations, compensate for pipetting error by making a 10.5X final staining solution).
   2. Add 10X worth of surface staining volume to the barcoded cell pellet. To reduce cell loss, with the same tip, mix and measure up the volume of surface stains and cell pellets.
   3. Based on the volume of cells and staining cocktail, add CSM to a final staining volume of 50 μL per number of cell samples (i.e., if running a set of 20 samples, 50 μL X 20 = 1 mL). Incubate for 30 min at 4 °C. Agitate the sample every 15 min to promote even staining.
   4. During the surface stain incubation, prepare the Perm/Wash buffer (Table of Materials) by diluting 1:10 with filtered ddH₂O. Prepare volumes of 2 mL for permeabilization and 5 mL for washing. Keep at 4 °C or on ice.
   5. Top off the surface staining tube with CSM, and centrifuge at 600 x g at 4 °C for 5 min. Aspirate the supernatant. Resuspend the barcoded sample in 2 mL of 4 °C, 1:10 dilution Perm/Wash buffer. Incubate at 4 °C for 20–30 min to fully permeabilize the cells.
   6. Centrifuge at 600 x g at 4 °C for 5 min and aspirate the supernatant.
   7. For intracellular staining, follow similar staining steps (as for surface staining). However, use the 4 °C, 1:10 dilution Perm/Wash buffer instead of the CSM to make the total staining volume so that the cells remain in a permeabilizing environment throughout the intracellular staining.
   8. Add the intracellular antibody cocktail to the surface stained and permeabilized cell pellet (steps 2.1.2–2.1.7). Bring the total staining volume to 50 μL per number of cell samples. Incubate for 60 min at 4 °C. Agitate the sample every 15 min to ensure even staining.
   9. During the intracellular staining, prepare the intercalator solution: 900 μL of filtered PBS + 100 μL of filtered 16% PFA (final concentration of 1.6% PFA) + 0.2 μL of 500 uM of intercalator dye (Table of Materials).
   10. Top off the cell pellet + intracellular antibody cocktail with cold 1:10 Perm/Wash buffer.
   11. Centrifuge at 600 x g at 4 °C for 5 min, and aspirate the supernatant. Top off the staining tube with CSM. Count and record the cell number. Centrifuge at 600 x g at 4 °C for 5 min, and aspirate the supernatant. Resuspend the cells in 1 mL of intercalator solution from step 4.9. Incubate for at least 20 min at room temperature for full intercalation or overnight at 4 °C (samples in intercalator solution can stay at 4 °C for up to 1 week before running them on the mass cytometry instrument).
2. Prepare the cells for the mass cytometry instrument.
   1. Top off the tube with 3 mL of filtered ddH₂O, and centrifuge at 600 x g for 5 min at 4 °C. Resuspend the cells in 3 mL of filtered ddH₂O. Pass this suspension through a 100 μm filter to remove any debris or aggregates that could potentially clog the mass cytometry instrument.
   2. Count and record the cell number post-filtration. Centrifuge at 600 x g for 5 min at 4 °C
3. Prepare the calibration bead solution (Table of Materials) by diluting it 1:10 in filtered ddH₂O.
4. Resuspend the stained cells in the required volume of 1:10 diluted calibration bead solution to attain a cell concentration of ~1 x 10⁶ cells/mL.
   Note: For a CyTOF1 at 45 µL/min, the recommended optimum is 5 x 10⁵ cells/mL; for Helios at 30 µL/min, 7.5 x 10⁵ cells/mL.
5. Proceed to run the sample on the mass cytometry instrument.