Rh103 intercalator

Single cell suspensions were stimulated with PMA (1:2000 of 100μg/mL stock, Sigma), ionomycin (1:1000 of 1mg/mL stock, Sigma), and GolgiStop (1:1500, BD Biosciences) x4hr. Cells were stained with Rh103 intercalator (500μM, Fluidigm) to label non-viable cells. Samples were stained with CyTOF antibody panels (Fig S1A, Table S2). Following staining, samples were labeled with Ir191/193 intercalator (1:1000 of 125μM stock, Fluidigm), then acquired on a Helios2 Mass Cytometer (Fluidigm). EQ 4 Elemental beads (Fluidigm) were used for normalization. Specifics of total cell numbers and viability of mucosal cells acquired in Table S3. Further details regarding CyTOF staining protocol can be found in Supplemental Methods.

Tissue cell suspensions were first incubated with Rh103 intercalator (Fluidigm, San Francisco, CA) for 20 min at 37°C to label nonviable cells, and then washed and labeled with a panel of metal-labeled antibodies for 30 min on ice. The samples were then fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) and incubated in 0.125uM Ir intercalator (Fluidigm) diluted in PBS containing 2% formaldehyde for 30 min. The samples were then washed and stored in PBS containing 0.2% BSA at 4°C until acquisition. Immediately prior to acquisition, samples were washed once with PBS, once with de-ionized water and then resuspended at a concentration of 1 million cells/mL in deionized water containing a 1:20 dilution of Equation 4 Element Beads (Fluidigm). The samples were acquired on a CyTOF2 (Fluidigm) equipped with a SuperSampler fluidics system (Victorian Airships) at an event rate of < 500 events/second. FCS files were manually pre-gated on Ir193 DNA⁺ events, excluding dead cells, doublets, and DNA^- negative debris. Samples were analyzed using Cytobank (Mountain View, CA).

CyTOF analysis was performed as previously described (Martin et al., 2019 (link)). Briefly, cell suspensions were incubated in Rh103 intercalator (Fluidigm; 1:500) at 37°C for 20 min to label nonviable cells. Cells were washed and then stained with a panel of CyTOF antibodies for 30 minutes on ice. Cells were then fixed in 1.8% isotonic PFA with 250 nM Ir (Fluidigm) with .25% OsO4 for 30 min before stored in PBS containing 0.2% BSA at 4°C until acquisition. Immediately prior to acquisition, samples were washed with PBS and de-ionized water and resuspended to 1 million cells/mL in deionized water with a 1:20 dilution of EQ Four Element Beads (Fluidigm). The samples were acquired on a CyTOF2 (Fluidigm) equipped with a SuperSampler fluidics system (Victorian Airships) at an event rate of < 500 events/second. Gating analysis was performed using Cytobank (Mountain View, CA).