Maxpar reagent

High-throughput mass cytometry (cytometry by time of flight (CyTOF)) analysis was performed as previously described.14 (link) Briefly, tumors were prepared as single-cell suspensions. Cells were pooled (3×10⁶) and immunostained with a mixture of metal-tagged antibodies using the different surface markers as indicated in online supplemental table S4. All antibodies were conjugated using the MAXPAR reagent (Fluidigm, South San Francisco, CA, USA) and tittered prior to staining. Rhodium and iridium intercalators were used to identify live/dead cells. Cells were washed twice with PBS, fixed in 1.6% formaldehyde (Sigma-Aldrich), washed again in ultrapure H₂O, and acquired by CyTOF mass cytometry system (Fluidigm). The acquired data were uploaded to the Cytobank web server (Cytobank). CD11b+ myeloid live cells were used for the analysis, and the gated cells were segregated into subpopulation clusters by expression markers. Data analysis was performed by viSNE algorithm,15 (link) via the Cytobank server. Changes in specific populations were validated by flow cytometry.

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.