A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

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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.

자료

Name	Company	Catalog Number	Comments
Ruxolitinib	Santa Cruz	SC-364729A	Stock Conc: 10 mM; Final Conc: 5 μM
R848	Invivogen	tlrl-r848-5	Stock Conc: 1 μg/μL; Final Conc: 1 μg/mL
LPS-EK	Invivogen	tlrl-eklps	Stock Conc: 1 μg/μL; Final Conc: 0.1 μg/mL
Sterile PBS	Lonza	17-516F
Lyse/Fix Buffer	BD biosciences	558049	Stock Conc: 5X; Final Conc: 1X (dilute in ddH2O)
BD Phosflow perm/wash buffer I	BD biosciences	557885	Stock Conc: 10X; Final Conc: 1:10 (dilute in ddH2O)
RPMI	Gibco	21870-076
Sodium Azide (NaN3)	Sigma-Aldrich	S-8032	Stock Conc:>99.9%; Final Conc: 0.0002
Protein Transport Inhibitor (PTI)	eBiosciences	00-4980-93	Stock Conc: 500X; Final Conc: 1X
DNA Intercalator	Fluidigm	201192B	Stock Conc: 500 μM; Final Conc: 0.1 μM
MaxPar Barcode Perm Buffer	Fluidigm	201057	Stock Conc: 10X; Final Conc: 1X
20-plex Pd Barcode Set	Fluidigm	S0014	Stock Conc: n/a; Final Conc: n/a
Antibodies used for Mass Cytometry
Surface markers
CD1c	Biolegend	L161	Mass: 161
CD3	BD	UCHT1	Mass: 144
CD4	Biolegend	SK3	Mass: 174
CD7	BD	M-T701	Mass: 149
CD8	Biolegend	SK1	Mass: 142
CD11b	Fluidigm	ICRF44	Mass: 209
CD11c	BD	B-ly6	Mass: 152
CD15	BD	HI98	Mass: 115
CD14	Biolegend	M5E2	Mass: 154
CD16	eBioscience/Thermo	B73.1	Mass: 165
CD19	Santa Cruz	SJ25C1	Mass: 163
CD21	Biolegend	Bu32	Mass: 141
CD27	BD	L128	Mass: 155
CD38	Fluidigm	HIT2	Mass: 172
CD45 total	Biolegend	HI30	Mass: 89
CD45RA	Biolegend	HI100	Mass: 153
CD56	Miltenyi	REA196	Mass: 168
CD66	BD	B1.1/CD66	Mass: 113
CD86	Fluidigm	IT2.2	Mass: 150
CD123	Fluidigm	6H6	Mass: 143
CD278/ICOS	Biolegend	C398.4A	Mass: 156
CD179/PD1	Biolegend	EH12.2H7	Mass: 162
IgD	Biolegend	IA6-2	Mass: 146
IgM	Biolegend	MHM-88	Mass: 151
CXCR5	BD	RF8B2	Mass: 173
HLADR	Biolegend	L243	Mass: 167
Cytokines
IL-1α	Biolegend	364-3B3-14	Mass: 147
IL-1β	Biolegend	H1b-98	Mass: 169
IL-1RA	Santa Cruz	AS17	Mass: 157
IL-6	Biolegend	MQ2-13A5	Mass: 164
IL-8	BD	E8N1	Mass: 160
IL-12/IL-23p40	Biolegend	C8.6	Mass: 171
IL-17A	Biolegend	BL168	Mass: 148
IL23p19	eBioscience/Thermo	23dcdp	Mass: 176
MIP1β	BD	D21-1351	Mass: 158
MCP1	BD	5D3-F7	Mass: 170
IFNα	Miltenyi	LT27:295	Mass: 175
IFNγ	Biolegend	4S.B3	Mass: 165
PTEN	BD	A2B1	Mass: 159
TNFα	Biolegend	Mab11	Mass: 166
Note: If the manufacturer is stated as Fluidigm, this antibody was purchased from Fluidigm with metal pre-conjugated. If the manufacturer is stated as other than Fluidigm, this antibody was self-conjugated using the MaxPar Multi-Metal Labeling Kit (Fluidigm Cat: 201300) according to manufacturer protocol.