Cellcapture software

Intracellular staining was performed on fixed and permeabilized RMS and RT cell suspensions with Transcription Factor Buffer Set (BD Biosciences, Jose, CA) prior to staining with fluorochrome-conjugated anti-human antibodies following manufacturer's protocol with minor modifications. Briefly, cells were incubated in Fix/Perm buffer for 40 min, washed with Perm/Wash buffer, and incubated for 15 min with 2% rabbit/2% mouse normal sera to block nonspecific binding. Cells were then stained for 2 hrs with mixture of fluorochrome-conjugated antibodies in Perm/Wash buffer: Bcl-2 (clone 100) from BioLegend, MYOD1 (polyclonal) and NOG (polyclonal) from Bioss Antibodies, MYOD1 (clone SPM427) and myogenin (clone MGN185 + F5D) from Novus Biologicals, and ID1 (clone B-8) from Santa Cruz Biotechnology, followed by staining with PE-streptavidin (Invitrogen) if biotinylated antibody was included in the staining mixture. The optimal antibody dilution was established by titration curve. Matching isotype controls were prepared as per standard flow cytometry protocols to determine background signals. All steps were performed on ice and samples kept in the dark. Flow cytometry was performed using an 8-color Stratedigm (San Jose, CA) S1000EX apparatus. Data were analyzed using CellCapTure software (Stratedigm).

Cells were incubated for 20 min on ice with 2 uL of anti-CD16/32 TruStain fcX (BioLegend, San Diego, CA) plus 10 uL of Brilliant Stain Buffer Plus (BD Biosciences, Franklin Lakes, NJ). Next, fluorochrome-labeled mAb were added, and the cells were incubated in the dark at RT for 20 min then were washed twice with 3 mL FACS buffer (1x PBS + 0.5% BSA + 0.1% NaN₃). Cells were then fixed with 200 uL IC Fixation buffer (Life Technologies Corp.) at RT for 20 min in the dark, then washed again with 3 mL FACS buffer, and stored at 4°C in the dark until analyzed. Flow cytometry was performed on a Stratedigm S1200Ex (Stratedigm Inc, San Jose, CA) and results were analyzed with CellCapTure software (Stratedigm).

Human ICAM‐1/CD54 Ab (0.5 µg; R&D Systems, Minneapolis, MA, USA) followed by a secondary FITC‐conjugated goat anti‐mouse Ab (1 : 50; Jackson ImmunoResearch Laboratories) was used for ICAM‐1 detection using flow cytometry as previously described [22]. Antigen expression was determined using Flow Cytometer S100EXi (Stratedigm; San Jose, CA, USA) with cellcapture software (Stratedigm, Inc., San Jose, CA, USA) and flowjo v10 (BD biosciences, Ashland, OR, USA). Dead cells were gated out from the analysis.

Cells were incubated on ice for 30 min with 2 μL of anti-CD16/32 TruStain fcX (BioLegend, San Diego, CA) plus 10 μL of Brilliant Stain Buffer Plus (BD Biosciences, Franklin Lakes, NJ). Next, fluorochrome-labeled mAb were added, and the cells were incubated at RT in the dark for 30 min then were washed twice with 3 mL FACS buffer (PBS + 0.5% BSA + 0.1% NaN₃). Cells were post-fixed with 200 μL IC Fixation buffer (Life Technologies Corp.) for 30 min at RT in the dark, then washed again with 3 mL FACS buffer, and stored at 4 °C in the dark until analyzed. Flow cytometry was performed on a Stratedigm S1200Ex (Stratedigm Inc, San Jose, CA) and analyzed with CellCapTure software (Stratedigm).
Intravascular leukocytes were distinguished from those located in brain parenchyma (extravascular) as previously described [39 (link)]. Briefly, mice were anesthetized with isoflurane then injected i.v. into the retroorbital sinus with 3 μg PE/Dazzle™-labeled anti-CD45 mAb (30-F11) in 50 μL of sterile 1× Dulbecco’s phosphate-buffered saline (DPBS). After 5 min, the mouse was euthanized by CO₂ asphyxiation and exsanguinated. After tissue preparation, leukocytes were incubated with PE-Cy7-labeled CD45 mAb, as well as other markers. Using this technique, CD45⁺ cells bearing PE/Dazzle were considered intravascular, whereas those not bearing this label were deemed extravascular.

One milliliter of logarithmic cells were washed with PBS buffer and sonicated using 15 s pulses at 20% power. For each sample, 2*10⁶ cells were resuspended with ProteoStat dye (Enzo Life Sciences) diluted 1:3000 in ProteoStat assay buffer. Cells were incubated for 15 min at room temperature protected from light. Flow cytometry was preformed using Stratedigm S1000EXi and the CellCapTure software (Stratedigm, San Jose, CA). Live cells were gated (P1) by forward scatter and side scatter. Fluorescence channels for FITC (530/30) and PE-Cy5 (676/29) were used utilizing a 488 nm laser source. A total of 50,000 events were acquired for each sample. Analyses were performed using FlowJo software (TreeStar, version 10). The results displayed are representative of three biological experiments performed in triplicate.

Cell surface expression of PD-L1 was determined by flow cytometry using mouse IgG1 antibodies against human PD-L1 (#14-5983-82, Thermo Fisher Scientific, Waltham, MA, USA), followed by FITC-conjugated goat anti-mouse IgG antibodies (#115-095-003, Jackson ImmunoResearch Laboratories). Baseline staining was determined by isotype-matched control mouse IgG antibodies (#400102; Biolegend, San Diego, CA, USA). Fluorescence was determined by Flow Cytometer S100EXi (Stratedigm, San Jose, CA, USA), using CELLCAPTURE software (Version 1; Stratedigm), and analyzed by FLOWJO V10 (Version 10.7.1; BD Biosciences, Franklin Lakes, NJ, USA).