Mass cytometry immunostaining protocol for multiplexing clinical samples.

This is a cytometry by time-of-flight (CyTOF) staining protocol for hematopoietic-derived cells, that leverages live-cell barcoding using receptor-type tyrosine-protein phosphatase C (CD45) antibodies conjugated to metal isotopes in combination with DNA-based palladium barcoding to multiplex up to 40 samples. In this protocol, DNA-based barcoding is performed before surface and intracellular immunostaining, which reduces the batch effects that result from day-to-day variations in staining and instrument sensitivity. This protocol also reduces antibody consumption and eliminates the need for repeated instrument adjustment.

Before you begin

This CyTOF staining protocol describes the steps for barcoding and staining human immune cells, including peripheral blood mononuclear cells (PBMCs) and single-cell suspensions isolated from human tissues and fluid samples, using titrated metal-tagged antibodies.

Institutional approvals

Ensure that all institutional approvals have been obtained prior to working with human specimens. This protocol includes examples using data generated from a human peripheral blood leukopak (institutional approval #56878).

Key resources table

Materials and equipment

Naturally abundant cis-platin can be used as an alternative to Cell-ID intercalator-Rh to label dead cells. However if used, isotopically pure platinum conjugated antibodies cannot be included. In addition, cis-platin is a known carcinogen/mutagen and if used should be handled with care.

Once opened, 16% formaldehyde should be stored at −20°C in 0.5 mL or 1 mL aliquots. Thaw aliquots as much as needed for the experiment. Thawed aliquots or leftover should not be refrozen and used.

Step-by-step method details

The day before staining

We recommend performing experiments over two consecutive days, as the protocol is lengthy and could be exhausting. In the first day, samples, buffers, regents, and labels are prepared as follows.

Timing: 2–3 h

Collect sample vials in a separate box for quick retrieval on the day of staining. Keep samples in liquid nitrogen storage until ready to begin the protocol. Cells were cryopreserved as described by Bull et al. (2007).

Prepare tubes, labels, buffers, and antibody mixtures as described below.

Fill pre-labeled 15 mL tubes with 5 mL of pre-warmed thawing media – prepared as described below. This is for resuspending the thawed samples. The number of tubes depends on the number of samples to be stained (up to 40).

Prepare antibody master mixes as described in step 2 below and store them at 4°C.

Prepare the barcode table, where each sample will be assigned to one CD45- Pt (either 194Pt or 198Pt) and one palladium code from the Cell-IDTM 20-plex Pd barcoding kit, as shown in Table 1.

Table 1

Barcodes table

Sample#	CD45	Barcode ID	Sample ID
1	194-Pt	Barcode 1
2	194-Pt	Barcode 2
3	194-Pt	Barcode 3
4	194-Pt	Barcode 4
5	194-Pt	Barcode 5
6	194-Pt	Barcode 6
7	194-Pt	Barcode 7
8	194-Pt	Barcode 8
9	194-Pt	Barcode 9
10	194-Pt	Barcode 10
11	194-Pt	Barcode 11
12	194-Pt	Barcode 12
13	194-Pt	Barcode 13
14	194-Pt	Barcode 14
15	194-Pt	Barcode 15
16	194-Pt	Barcode 16
17	194-Pt	Barcode 17
18	194-Pt	Barcode 18
19	194-Pt	Barcode 19
20	194-Pt	Barcode 20
21	198-Pt	Barcode 1
22	198-Pt	Barcode 2
23	198-Pt	Barcode 3
24	198-Pt	Barcode 4
25	198-Pt	Barcode 5
26	198-Pt	Barcode 6
27	198-Pt	Barcode 7
28	198-Pt	Barcode 8
29	198-Pt	Barcode 9
30	198-Pt	Barcode 10
31	198-Pt	Barcode 11
32	198-Pt	Barcode 12
33	198-Pt	Barcode 13
34	198-Pt	Barcode 14
35	198-Pt	Barcode 15
36	198-Pt	Barcode 16
37	198-Pt	Barcode 17
38	198-Pt	Barcode 18
39	198-Pt	Barcode 19
40	198-Pt	Barcode 20

Preparing buffers and reagents.

CyTOF staining media (CSM): sterile PBS supplemented with 2% FBS and 0.5% sodium azide. Store at 4°C.

Thawing media: RPMI containing 5% FBS. Incubate in 37°C water bath.

Preparing antibody master mixes.

∗Prior to preparing staining master mixes, subtract the total volume of antibodies that will be added from the desired final volume of master mix to determine the amount of diluent (CSM or permeabilization buffer) to use.

Surface stain – master mix 1: Prepare a 2× mix in CSM including all the pre-titrated, pre-conjugated extracellular antibodies in Table 2.

Table 2

Dilution of extracellular antibodies

Tag	Marker	2× dilution	Antibody volume (ul)
141 Pr	CD45RA	200	1.75
112 Cd	CD45RO	200	1.75
152 Sm	CD11c	200	1.75
158 Gd	CD27	200	1.75
159 Tb	CD19	200	1.75
162 Dy	CD28	200	1.75
172 Yb	CD127	200	1.75
142 Nd	HLA-DR	100	3.5
144 Nd	CD163	100	3.5
146 Nd	CD8	100	3.5
147 Sm	CD4	100	3.5
151 Eu	CD39	100	3.5
154 Sm	CD69	100	3.5
156 Gd	CD14	100	3.5
160 Gd	CD25	100	3.5
165 Ho	PD1	100	3.5
168 Er	CD73	100	3.5
173 Yb	NKp46	100	3.5
175 Lu	PDL1	100	3.5
166 Er	TIM3	100	3.5
153 Eu	CD3	50	7
111 Cd	CD80	50	7
143 Nd	ICOS	50	7
148 Nd	CD15	50	7
150 Nd	CD103	50	7
155 Gd	CD303	50	7
163 Dy	CD137	50	7
164 Dy	CD86	50	7
169 Tm	TCRgd	50	7
176 Yb	LAG3	50	7
209 Bi	CD16	50	7
	Total volume of Abs		134.75
	CSM volume		40.25

Prepare a 1.5 mL Eppendorf tube with the volume of CSM necessary to achieve a 2× antibody concentration. Place the individual antibody vials and the Eppendorf on ice. Arrange the vials in the order of the antibody list.

Add the first antibody to the Eppendorf tube to reach the desired concentration. A final volume of 50 μL is sufficient for staining up to 10 × 106 PBMCs. For highly multiplexed samples, a master mix volume of 150 μL is recommended. Prepare 25 μL of extra master mix to account for any pipettor error.

For example, to make a 2× anti-CD3 that has an optimal dilution of 1:50 in a final volume of 175 μL, add 7 μL of conjugated anti-CD3 antibody to 40.25 μL of CSM. Add the rest of the antibodies similarly. See Table 2.

Once added, transfer the vial to another ice bucket and check it off of the antibody list. This helps keep track of the antibodies that have been added and prevents errors in repetition or omission.

Repeat steps 2.1.2 and 2.1.3 until all surface antibodies have been added.

Store master mix 1 at 4°C and vortex before use.

Intracellular stain – master mix 2: Prepare a 1× mix in 1× permeabilization buffer according to the pre-determined optimal dilutions for each of the intracellular antibodies in Table 3.

Table 3

Dilution of Intracellular antibodies

Tag	Marker	2× dilution	Antibody volume (ul)
113 ln	IL-10	200	1.625
116 Cd	Ki67	200	1.625
170 Er	CTLA4	200	1.625
171 Yb	Granzyme B	100	3.25
114 Cd	Helios	50	6.5
115 ln	SOCS1	50	6.5
149 Sm	FOXP3	50	6.5
161 Dy	T-bet	50	6.5
167 Er	PKR	50	6.5
174 Yb	TCF-1	50	6.5
	Total volume of Abs		47.125
	Permeabilization buffer		277.875

A final volume of 100 μL is sufficient for staining up to 10 × 106 PBMCs. For highly multiplexed samples, a master mix volume of 300 μL is recommended. Prepare 25 μL of extra master mix to account for any pipettor error.

Prepare a 1.5 mL Eppendorf tube as in 2.1.1 with the volume of permeabilization buffer needed to achieve a 1× concentration.

Add antibodies as in 2.1.2–2.1.4.

Store master mix 2 at 4°C and vortex before use.

A- The optimal dilutions of all the metal-tagged antibodies used in these protocol were determined through serial dilutions testing as described by van Vreden et al. (2019).

B- We highly recommend that the performance of the panel should be examined first on control samples prior to proceeding to valuable clinical samples.

On day of staining

The major steps on the second day are staining cells with viability marker, surface and intracellular markers to be able to identify cell types and protein expression.

Timing: 6 h

Prepare the following buffers. Store at 4°C and vortex before use.

1× Maxpar perm barcode buffer: dilute 10× Maxpar barcode buffer using Maxpar PBS.

Fixation/permealization buffer: add fixation/permeabilization concentrate from eBioscience FOXP3/transcription factor staining buffer kit to the fixation/permeabilization diluent at dilution 1:3 (e.g., 100 μL of the fixation concentrate to 300 μL of the dilutent).

Fc blocker: add 50 μL of Fc receptor binding inhibitor to 450 μL CSM.

Viability buffer: add 8 μL Cell-ID™ intercalator – 103Rh to 4 mL of RPMI.

1× Permeabilizaiton buffer: add 1 mL of 10× Permeabilization Buffer from eBioscience FOXP3/transcription Factor Staining Buffer Kit to 9 mL of ddH2O.

DNA staining buffer: prepare 1 mL of iridium-DNA intercalation solution by adding 1 μLCell-ID™ intercalator-Ir (125 μM) to 1 mL of Maxpar fix and perm buffer.

Antibody master mix filtration:

Add each antibody master mix into a separate 0.1 μm Millipore centrifuge tube.

Spin the centrifuge tubes with a fixed-angle centrifuge rotor at 12,000 × g, 4°C for 2 min.

Discard the Millipore filter and store the tubes at 4°C.

Thawing cells:

Bring a bucket filled with dry ice and another bucket filled with regular ice to the 37°C water bath.

Place sample vials on the dry ice and place the pre-labeled 15 mL tubes in the ice bucket.

Thaw samples one at a time by placing vial on a rack in the 37°C water bath for approximately 1 min.

Quickly transfer the contents of the freshly thawed vial into the appropriate pre-labeled tube filled with 5 mL thawing media and leave it on ice.

Centrifuge the sample tubes at 600 × g, 4°C for 5 min.

Aspirate the supernatant.

If the samples cell count were not previously performed and recorded prior to cryopreservation, count each sample manually using a hemocytometer and record the viable cell counts.

Resuspend cell pellets in 100 μL thawing media again and transfer it to a 96-well V-bottom plate∗.

∗Notes:

A- Ideally, reserve up to 2 million cells from each sample for staining. Expect ∼30%–50% cell loss during the staining process. Note, we have stained as few as 10,000 cells from a single sample (Gadalla et al., 2019).

B- Arrange samples in the plate so that there are 10 samples in each row, and each barcode set is separated from the other to avoid mixing up staining (Figure 1A).

Figure 1

Sample and Maxpar barcoding kit vial layouts

(A) Figure shows the layout of samples in a 96-well V-bottom plate.

(B) Picture shows Maxpar barcoding kit minivial strips.

i. Centrifuge the plate at 600 × g, 4°C for 3 min. Discard supernatant.

Viability stain and live-cell barcoding:

For 40 samples experiment, split the 4 mL of 103Rh viability buffer in two 2 mL Eppendorf tubes.

Add 40 μL of CD45-194Pt to one of the 103Rh-viability buffer tubes (dilution 1:50), and 40 μL of CD45-198Pt to the other 103Rh-viability buffer tube. These antibodies were pretitrated for optimal performance.

Other metal-tags for CD45 antibodies could be used. 194Pt and 198Pt here are just examples. This need to be tailored according to each panel and the available channels.

Using multichannel pipette, add 100 μL of the CD45-194Pt-103Rh-RPMI mix to the first 20 wells of the 96-well V-bottom plate containing your samples, and mix by gentle pipetting.

Add 100 μL of the CD45-198Pt-103Rh-RPMI mix to the remaining 20 wells of the 96-well V-bottom plate containing the rest of your samples, and mix by gentle pipetting.

Put the cover lid on the 96-well V-bottom.

Incubate the plate for 15 min at 37°C.

Add 100 μL of PBS to each well.

Centrifuge at 600 × g, 4°C for 3 min.

Flick the plate to discard the supernatant.

Blot the plate gently on paper towel to get rid of any residual supernatant.

Fixation and permeabilization:

Add 5 mL of the previously prepared fixation/permeabilization buffer to a multichannel pipette solution basin.

Using a multichannel pipette, add 100 μL of fixation/permeabilization buffer to each well and resuspend cells by gentle pipetting.

Incubate for 15 min at room temperature.

Remove 2 sets of barcode vials from the −20°C freezer (each set has 20 mini-vials labeled from 1 to 20 – each 10 barcodes are attached in one single strip).

Place the 4 mini-vial strips in rack as shown in Figure 1B and let them thaw at room temperature for the next step.

Add 100 μL of 1× Maxpar perm barcode buffer to each well of the plate containing the sample using multichannel pipette and solution basin.

Centrifuge the plate at 900 × g, 4°C for 3 min.

Flick the plate to discard the supernatant.

Blot the plate gently on paper towel to get rid of any residual supernatant.

Barcoding:

Add 100 μL of 1× Maxpar barcode buffer to each barcode mini-vial, and mix well by gentle pipetting.

Transfer 100 μL from each barcode vial to its respective sample well in the 96-well V-bottom plate according to the barcode map (Table 1). Mix well by gentle pipetting.

Incubate for 30 min at room temperature.

Add 100 μL of Maxpar CSM to each well.

Centrifuge at 900 × g, 4°C for 3 min.

Flick the plate to discard the supernatant.

Blot the plate gently on paper towel to get rid of any residual supernatant.

Add 200 μL of Maxpar CSM to each well. Repeat steps 7.5 and 7.6.

∗Washing the cells twice ensures that no leftover barcodes remain in any wells, and prevents mixing of barcodes when the samples are pooled in the next step.

Add 100 μL Maxpar CSM to each well.

Pool samples by transferring the content of each well to a 4 mL polystyrene conical “FACS” tube.

Centrifuge at 900 × g, 4°C for 5 min.

Aspirate and discard the supernatant.

Cell staining:

Fc blocking step:

For up to 80 × 106 cells, resuspend cells in 150 μL of Fc blocker and mix by gentle pippetting.

Incubate for 10 min at room temperature.

Surface staining:

Add 150 μL of 2× surface stain (master mix 1) directly to the sample (suspended in same volume of Fc blocking buffer) to make final 1× concentration of antibodies cocktail. Mix by gentle pipetting.

Incubate for 30 min at 4°C.

Add 2 mL of CSM to each well, and mix by gentle pipetting.

Centrifuge at 900 × g, 4°C for 5 min.

Add 2 mL 1× permeabilization buffer and resuspend cell pellet by gentle pipetting.

Centrifuge at 900 × g, 4°C for 5 min.

Aspirate and discard the supernatant.

Intracellular staining:

Add 300 μL of 1× intracellular stain (master mix 2); mix by pipetting up and down 3 times and incubate for 30 min at 4°C.

Add 2 mL of PBS.

Centrifuge at 900 × g, 4°C for 5 min.

Aspirate and discard the supernatant.

Iridium labeling of cellular DNA:

Suspend cell pellet in 300 μL of DNA staining buffer.

Incubate for 30 min 4°C.

Add 2 mL PBS.

Centrifuge at 900 × g, 4°C for 5 min.

Aspirate and discard the supernatant.

Storing stained cells:

Samples can be stored in 200 μL of 1.6% FA/PBS for up to 1 week at 4°C.

∗For long-term storage, cryopreservation in 10% DMSO 90% FBS has been shown to be effective in preserving the signal quality for up to 4 weeks (Sumatoh et al., 2017).

Preparing stained cells for acquisition.

On the day of acquisition, add 2 mL of PBS to the stained and barcoded cells.

Centrifuge at 900 × g, 4°C for 5 min.

Aspirate the supernatant and resuspend the cells in 1 mL PBS.

Count cells manually using a hemocytometer and record the cell count.

Obtaining the total cell count is necessary for CyTOF acquisition, as the CyTOF operator uses it to determine the volume of the acquisition buffer mixed with normalization beads to add to the sample (Lee and Rahman, 2019).

Filter the cells using the 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap.

Centrifuge at 900 × g, 4°C for 5 min.

Discard supernatant and keep the tube containing the cell pellet on ice.

More than 40 samples can be barcoded and stained by using more than two CD45 antibodies e.g., 60 samples can be barcoded by using 3 separate CD45 antibodies (Thrash et al., 2020).

Expected outcomes

This protocol details all of the necessary steps prior to sample acquisition on the Helios mass cytometer. Acquired data are saved as FCS files. The data are exported in a bead-normalized FCS file format that is ready for downstream analysis, including data preparation, and debarcoding to separate individual samples. Figure 2 shows the use of Gaussian parameters to exclude unwanted events/noise from the data (Bagwell et al., 2020). Figure 3 shows the use of biaxial plotting to debarcode the data based on combination of the platinum and palladium signals Individual cells are assigned to specific barcodes based on the absent and presence of platinum and palladium signals (Mei et al., 2015).

Figure 2

Standard CyTOF data preparation steps

(A) Normalization beads added during acquisition are excluded by plotting time vs Ce-140.

(B) Series of biaxial plots of time vs Gaussian parameters (residual, center, offset, width) used to eliminate non- specific signals.

(C) Plot shows time vs event length, which is used to eliminate signals from cell aggregates.

(D) Plot shows time vs 103Rh to distinguish live and dead cells.

(E) Plot shows time vs DNA-Ir191 and Ir193 to eliminate cell debris and to gate on intact cells. Using time as a common parameter in all plots is important to monitor signal fluctuations during acquisition. These steps can be performed using any software that is compatible with FCS-based cytometry data e.g., FlowJo, CytoBank.

Figure 3

Manual debarcoding strategy

Manual debarcoding through a series of gates on biaxial plots aims to separate the individual samples by sequentially deconvoluting the CD45 barcodes and DNA-palladium barcodes.

(A) Biaxial plot with gates on samples stained with each of the CD45 antibodies.

(B) Barcode map from Cell-IDTM 20-plex Pd barcoding kit, where rows represent the barcode (sample number) and columns represent the associated palladium isotope mass number.

(C and D) (C) Plots show examples of debarcoding gates for samples number 1 and 2, and (D) samples number 21 and 22. Note: This figure is not generated from single-experiment data. It was put together to show the workflow.

Typically, high-dimensional data analysis tools are employed with CyTOF data, such as phenograph-based clustering (Levine et al., 2015), t-Distributed Stochastic Neighbor Embedding (Amir et al., 2013), and Uniform Manifold Approximation and Projection for dimension reduction (UMAP) (Becht et al., 2018) to identify and visualize immune populations in different experimental groups and/or samples (Mathew et al., 2020; Lheureux et al., 2022).

Limitations

This protocol is designed for the analysis of CD45-expressing human immune cells. Therefore, only barcoded immune cell data will be acquired from any samples that also include non-immune cells, such as single-cell suspensions of tumor biopsies or ascites/pleural effusion fluids from cancer patients. If the non- immune components of the samples are of interest, then the same protocol can be used with DNA-based barcoding alone – without the use of multiple CD45 antibodies – and thus limiting the sample number to 20 per batch (Zunder et al., 2015; Lheureux et al., 2022).

Cell/event loss occurs at several points in the protocol: during repeated wash steps, cell filtering steps, cell acquisition, as well as during debarcoding. This should be taken into consideration when designing the experiment and choosing the number of cells to stain and acquire. Reducing the volume in the washing steps helps with reducing cell loss.

CyTOF data acquisition: the average and optimal rate of acquisition on the Helios mass cytometer is 300 cells/s (approximately 1 million cells/h). Higher acquisition rates could lead to clogging, increased numbers of doublets and aggregate events as a result of ion clouds overlapping. When barcoding up to 40 samples, and depending on the abundance of the immune population(s) of interest, long acquisition times (10–12 h) are expected.

Costs and panel design: there are many possible combinations of antibodies that can be used to create 40+ marker CyTOF panels, but commercially available ready-to-use antibodies are limited. Custom antibody-metal conjugations are an additional cost to consider.

Cells are not recoverable after acquisition:unlike traditional flow cytometry, where sorted population(s) of interest can be viably isolated for subsequent downstream applications (e.g., functional assays, RNA-sequencing), cells acquired by the CyTOF are vaporized.

Traces of platinum in biospecimens from patients who have been subjected to any form of platinum-based therapy, will interfere with the platinum isotope signal. In this case, other metal-tags, can be used for CD45-barcoding.

Troubleshooting

Problem 1

High level of 138Ba signal during acquisition.

Potential solution

Ambient barium is a known contaminant of CyTOF staining and can be avoided by keeping samples closed/covered, working in a laminar flow hood and changing out older buffers.

Problem 2

High background staining and/or high number of events recorded that do not correspond to cells.

This problem could indicate insufficient washing, poor antibody filtration, incorrect antibody concentration or an expired antibody. Ensure no wash steps are skipped. Always filter antibody mixes on the day of staining (not before). Re-titrate antibodies if necessary. Check antibody stock for black flecks (metal precipitates) that could indicate the antibody is no longer usable.

Problem 3

Low fraction of events corresponding to cells (191-Ir/103-Ir double positive). Potential solution:

See troubleshooting tips for Potential problem 2. Another possibility is suboptimal DNA staining. An alternative DNA staining buffer can be used with FA and 0.3% saponin (See materials and equipment setup). Alternatively, longer incubation periods and staining at room temperature can increase the fraction of labeled cells.

Problem 4

Signal spill-over from the platinum tags and/or high background in the platinum channels.

Normally, there is no signal spill-over from the platinum tags, if monoisotopic platinum is used to conjugate CD45. However, if this problem occurred, it could be a result of long incubation time or high antibody concentration. The combination of time and incubation will then have to be determined through testing various incubation times and antibody concentrations.

Problem 5

Time-consuming debarcoding.

Manual debarcoding consumes a lot of time. CyTOF software provided by Fluidigm has a debarcoding functionality, but it is dedicated to palladium barcodes only. So, users can debarcode the CD45 platinum tag and then input two separate files into Fluidigm software to debarcode the palladium tags. Alternatively, R package premessa allows users to define the barcodes in a graphical user interface. However, implementation of these methods requires users to set global parameters/thresholds that are not always accurate to all samples, which results in significant cell loss.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Ben Wang; ben.wang@uhnresearch.ca.

Materials availability

No newly generated materials or reagents are associated with this protocol.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CD16	Fluidigm	3209002B
CD80	BD Biosciences	55568
CD45RA	BioLegend	304143
Helios	BioLegend	137202
Ki67	BioLegend	350523
T-bet	Fluidigm	3161014B
CD45	Fluidigm	3089003B
CD45	Fluidigm	3198001B
CD28	BioLegend	302937
CD137	BioLegend	309802
CD86	BioLegend	555655
TIM3	BioLegend	345019
PKR	Novus Biologicals	Custom-made
CD73	Fluidigm	3168015B
CTLA4	Thermo Fisher Scientific	14-1529-82
CD39	BioLegend	328221
CD3	BioLegend	300443
CD303	BioLegend	354215
CD14	BioLegend	301802
CD27	BioLegend	302839
CD25	BioLegend	356102
PD1	Fluidigm	3165039D
IL-10	BioLegend	501423
SOCS1	Millipore	04-002
PDL1	Fluidigm	3175008B
HLA-DR	Fluidigm	307651
ICOS	BD Biosciences	557801
CD163	BioLegend	333602
CD8	BioLegend	301053
CD15	BioLegend	323002
CD103	Thermo Fisher Scientific	14-1038-82
CD4	BioLegend	300541
FOXP3	Thermo Fisher Scientific	14-4777-82
CD11c	BioLegend	337221
CD69	BioLegend	310939
CD19	BioLegend	302202
TCRgd	Thermo Fisher Scientific	TCR1061
Granzyme B	Thermo Fisher Scientific	MA1-80734
CD127	Thermo Fisher Scientific	14-1278-82
NKp46	BioLegend	331902
TCF-1	BD Biosciences	Custom-made
LAG3	BioLegend	369202
CD45RO	BioLegend	304239
Biological samples
Human Peripheral Blood Leukopak, Fresh	STEMCELL Technologies	70500
Chemicals, peptides, and recombinant proteins
Maxpar® 10× Barcode Perm Buffer	Fluidigm	201057
Maxpar® PBS	Fluidigm	201058
RPMI 1640 Medium	Gibco	11875093
Cell-IDTM Intercalator-103Rh 500 μM	Fluidigm	201103A
Cisplatin	BioVision	1550-1000
Cell-IDTM Intercalator-Ir-125 μM	Fluidigm	201192A
eBioscience FOXP3/Transcription Factor staining buffer kit	Thermo Fisher Scientific	00-5523-00
Maxpar® Fix and Perm Buffer—100 mL	Fluidigm	201067
PBS, Ca2+ and Mg2+-free, pH 7.2	Gibco	20012-050
16% Formaldehyde, methanol-free, Ultrapure	Analychem Corporation	18814-20
Albumin bovine serum, BSA	Sigma-Aldrich	12657-25GM
Fc Receptor Binding Inhibitor Polyclonal Antibody, Fc blocker	Invitrogen	16-9161-73
Critical commercial assays
Cell-IDTM 20-plex Pd Barcoding Kit	Fluidigm	201060
FlowJo v10	FlowJo	https://www.flowjo.com/solutions/flowjo/downloads
Cytobank	Cytobank Inc.	https://premium.cytobank.org
Other
0.1 μm pore size, hydrophilic PVDF, 0.5 mL volume	MilliporeSigma	UFC30VV25
12 × 75 mm Tubes, Polystyrene	Diamed	STK 8550
Falcon® 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap	Corning	352235
96 Well Conical V-Bottom Plate	Thermo Fisher Scientific	277143
Sterile Multichannel Disposable Solution Basins
Centrifuge
Recently calibrated P10, P20, P200, and P1000, and a P200 multichannel pipette
CO2 incubator
Hemocytometer
Biological safety cabinet
Vacuum aspiration system
Fluidigm Helios™ Mass Cytometer	Fluidigm

CyTOF staining media (CSM)

Reagent	Final concentration	Amount
FBS	2%	10 mL
PBS	N/A	490 mL
Sodium azide∗	0.5%	0.1 g
Total	N/A	500 mL

Store at 4°C.

Sodium azide is highly toxic if ingested or inhaled, and is a skin irritant. Use personal protective equipment when dispensing and only handle powder under a fume hood.

Alternative CyTOF Staining Media (CSM)

Reagent	Final concentration	Amount
BSA	1%	150 g
Sodium azide∗	0.5%	25 g
PBS	N/A	500 mL
Total	N/A	500 mL

Sodium azide is highly toxic if ingested or inhaled and is a skin irritant. Use personal protective equipment when dispensing and only handle powder under a fume hood.

Alternative DNA staining buffer

Reagent	Final concentration	Amount
Cell-ID™ intercalator-Ir(500 μM)	100 nM	1 μL
16% formaldehyde (frozen aliquots)	1.6%	0.5 mL
0.3% saponin (filtered 1.5 g in 500 mL PBS)	N/A	4.5 mL
Total	N/A	5 mL

Prepare fresh on day of staining.