Rhodium

Purified CNS-single cells (3 × 10⁶ per sample) were first incubated with FcR blocking reagent (cat. 130-092-575; Miltenyi Biotec) according to manufacturer’s protocol and then stained (100-μl final staining reaction volume; 1 h; 4 °C) with a mixture of metal-tagged anti-cytokine/chemokine/growth antibodies (a complete list of antibodies is provided in Additional table 1) conjugated using the MAXPAR reagent (Fluidigm Inc.). Rhodium (1:2,000; Fluidigm Inc.) was added to the cells in the last 20 min of staining. Cells were then washed twice with staining buffer, fixed in 1.6% PFA (Sigma–Aldrich) in PBS (1 h, RT), stained with iridium (1:2,000, 20 min, RT; Fluidigm Inc.), washed again in ultrapure H₂O (to prevent cell loss, the sample was centrifuged at 10,000 g for 1 min), and analyzed on a CyTOF I machine (Fluidigm Inc.), with events acquired at approximately 500 events per second. Internal metal-isotope bead standards were added for sample normalization. Acquired data were uploaded to a Cytobank webserver (Cytobank Inc.) for data processing and for gating out of dead cells and normalization of beads. Rhodium (Rh) and iridium (Ir) (Fluidigm Inc.) inter-chelators were used to identify live/dead cells. At least 20,000 live single cells were analyzed in each sample. The Rh gating was used to assess live/dead cells [29 (link)].

Total FL cells were collected and incubated with TruStain fcX (Biolegend) for FC blocking. After being washed twice with staining buffer (PBS containing 1% bovine serum albumin and 0.05% sodium azide), the FL cells were stained with a mixture of metal‐tagged antibodies (see Table S1, Supporting Information, for the complete antibodies list). All antibodies were conjugated using the MAXPAR reagent (Fluidigm Inc.). Rhodium (1:2000; Fluidigm Inc.) was added to the cells for the last 20 min of staining. Cells were fixed with 1.6% PFA (Sigma‐Aldrich) in PBS and stained with iridium (Fluidigm Inc.).The samples were analyzed on a CyTOF III machine (Fluidigm Inc.). Acquired data were processed using a Cytobank web server (Cytobank Inc.). Rhodium (Rh) and iridium (Ir) (Fluidigm Inc.) intercalators were used to identify live/dead cells.