Maxpar antibody labeling kit

Antibodies not purchased from Fluidigm were labeled using metal tags using Maxpar Antibody Labeling Kits (Fluidigm). Antibody titration was undertaken and used at a concentration from 0.25 to 0.5 μg/mL. One to three million cells were first stained per sample with a solution containing rhodium DNA intercalator (Fluidigm) to distinguish live/dead cells before Fc receptor blocking (Miltenyi Biotec). Samples were then stained with the mixture of conjugated antibodies for cell-surface antigens. After washing in Maxpar Cell Staining Buffer (Fluidigm), samples were fixed and permeabilized using the Foxp3 Transcription Factor Staining Buffer Kit (Thermo Fisher) prior to washing and incubation with metal-conjugated antibodies recognizing intracellular antigens. Samples were washed twice in cell-staining buffer, fixed by incubation with 1.6% paraformaldehyde (PFA) (Pierce) for 10 min, and finally incubated overnight with iridium DNA intercalator in Maxpar Fix and Perm Buffer (Fluidigm). Samples were washed again, being acquired on a Helios mass cytometer (Fluidigm). After acquisition, .fcs files were normalized using tools within Helios software, and gating and downstream analysis using clustering and dimensionality reduction algorithm viSNE was performed using Cytobank (https://www.cytobank.org/).

Lanthanide-conjugated antibodies were purchased from Fluidigm. Antibodies not available in metal-conjugated form were purchased in carrier-free solution and validated by brightfield immunohistochemistry using the appropriate isotype control antibodies. Subsequently, antibodies were conjugated to lanthanide metal isotopes following the Maxpar® Antibody Labeling Kit protocol (Fluidigm). Briefly, carrier-free antibodies were partially reduced with Bond-Breaker™ TCEP buffer (Thermo Scientific) at 37 °C before incubation with purified, lanthanide-loaded Maxpar® X8 polymer at 37 °C for 90 min. The percent yield of metal-conjugated antibodies was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Metal-conjugated antibodies were stored at 0.5 mg/mL in PBS-based Antibody Stabilizer (Candor Bioscience) with 0.05% sodium azide at 4 °C. Working concentrations for all metal-conjugated antibodies were optimized by IMC (Additional file 1: Table S2) on MS lesion tissue.

Antigens were selected based on previously published single-cell mass cytometry and single-cell RNA sequencing data on the human fetal intestinal samples (1 (link), 12 (link), 13 (link)). Antibodies used for IMC are listed in Table 1. 16 of the 34 antibodies used in the current panel were directly purchased from Fluidigm, which were already conjugated with metals. For the remaining 18 antibodies, BSA-free and carrier-free formulations of antibodies were purchased from different suppliers and initially tested for performance by immunohistochemical staining (IHC) on human fetal intestine and tonsil. Subsequently, antibodies with an appropriate signal intensity were conjugated to lanthanide metals using the MaxPar Antibody Labeling Kit (Fluidigm) following the manufacturer's instructions. Post-conjugation, all antibodies were eluted in 100 μl W-buffer (Fluidigm) and 100 μl antibody stabilizer buffer (Candor Bioscience, Wangen im Allgäu, Germany) supplemented with 0.05% sodium azide.

NLRP3 inhibitor tool compound MCC950 (CP-456773 sodium salt, PZ0280) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. Lipopolysaccharide (LPS) from Escherichia coli (O55:B5; ALX-581-013-L002) was obtained from Enzo. Nigericin (tlrl-nig-5) and ultrapure flagellins from Bacillus subtilis (Fla-BS; tlrl-pbsfla), Pseudomonas aeruginosa (Fla-PA; tlrl-pafla) and Salmonella typhimurium (Fla-ST; tlrl-epstfla-5) were obtained from Invivogen (purity >95% and endotoxin levels of <0.05 EU/µg). Propidium iodide (PI; P3566) and Lipofectamine 2000 (LF2000; 11668030) were obtained from ThermoFisher. Anti-mouse antibodies for mass cytometry were obtained either pre-conjugated to metal isotope or conjugated in-house using the Maxpar Antibody Labeling Kit (Fluidigm) following manufacturer’s instructions.

Metal-labeled antibodies used in this work, including manufacturer and staining-concentrations, are listed in Table 2. Unconjugated antibodies were metal tagged using the MaxPar Antibody Labeling kit (Fluidigm) according to the manufacturer’s protocol. Prior to use, the final concentration of each metal-labeled antibody was individually determined by titration and subsequent CyToF measurements. For long-term storage conjugated antibodies were diluted in antibody stabilization solution (Candor Bioscience GmbH) and stored at 4 °C.Table 2

Antibodies used for mass cytometry staining.

Number	Antibody	Clone	Metal	conc [µg/ml]
1	CD81	5A6	Yb173	0.12
2	CD169	7-239	Er170	0.25
3	CD32	FUN-2	Er166	0.25
4	HLA-ABC	W6/32	Yb172	0.25
5	PD-L1	29E.2A3	Gd160	0.25
6	CD304	12C2	Gd156	0.5
7	CD38	HIT2	Nd142	0.5
8	CD54	HA58	Yb176	0.5
9	CD82	ASL-24	Tb159	0.5
10	CD86	2331 (FUN-1)	Yb171	0.5
11	CD88	S5/1	Yb174	0.5
12	CD14	RMO52	Er168	1
13	CD163	GHI/61	Tm169	1
14	CD204	351615	Sm149	1
15	CD206	15-2	Sm147	1
16	CD64	10.1	Pr141	1
17	CD68	Y1/82 A	Nd143	1
18	CD71	CY1G4	Nd145	1
19	CD87	VIM5	Dy163	1
20	HLA-DR	L243	Lu175	1
21	CD119	GIR-208	Dy161	2
22	CD11b	M1/70	Eu151	2
23	CD120b	3G7A02	Dy164	2
24	CD123	6H6	Nd150	2
25	CD13	WM15	Nd148	2
26	CD155	SKII.4	Nd146	2
27	CD16	3G8	Er167	2
28	CD166	3A6	Gd158	2
29	CD197	GO43H7	Sm154	2
30	CD209	DCS-8C1	La139	2
31	CD282	TL 2.1	Ho165	2
32	CD36	5-271	Nd144	2
33	CD40	5c3	Sm152	2
34	CXCR4	12G5	Dy162	2
35	PD-L2	MIH18	Gd155	2
36	SLAMF7	162.1	Eu153	2

Metal-conjugated antibodies were either purchased from Fluidigm or coupled in-house with DN3 or X8 polymers using the MaxPar antibody labeling kit (Fluidigm). For palladium coupling, metal was added to anti-CD45 antibodies with amine-reactive isothiocyanobenzyl-EDTA chelator, as described by Mei and colleagues (79 (link)). See Supplementary Table 2 for staining panel and clone details.