Topcount nxt instrument

Single-cell suspensions from tumors were prepared as described above. Cells were stained and sorted on BD FACS Aria (Biosciences). Live M-MDSCs (CD11b^þ Ly6G^NEG Ly6C^HI) and PMN-MDSCs (CD11b^þ Ly6G^POS Ly6C^INT) were plated in U-bottom 96-well plates in RPMI with 10% FBS and cocultured 1:1 with splenocytes from PMEL transgenic mice in the presence of cognate peptides: PMEL (EGSRNQDWL; 0.1 mg/mL). After 48 hours, cells were incubated with [³H] thymidine (PerkinElmer) for 16 hours. Proliferation was measured using the TopCount NXT instrument (PerkinElmer). Positive controls were incubated consisting of PMEL transgenic splenocytes plus peptide and negative controls with the coculture in absence of peptide.

Radioligand competition binding assays were conducted with slight modifications as previously described by our laboratory.^{39 (link)} HEK293 cells stably transfected with human D1R (Codex Biosolutions) were dissociated from plates using EBSS-, and intact cells were collected by centrifugation at 900g for 10 min. Cells were resuspended and lysed using 5 mM Tris-HCl and 5 mM MgCl₂ (pH 7.4) at 4 °C. The cell lysate was centrifuged at 30000g for 30 min, and the membrane faction was resuspended in EBSS with calcium at pH 7.4. Cell membranes (100 μL, containing ~8 μg of protein for D2-like receptor assays or ~10 μg of protein for D1-like receptor assays) were incubated for 90 min at room temperature with either [³H]SCH23390 (D1R and D5R) or [³H]methylspiperone (D2R–D4R) in a final reaction volume of 250 μL. Nonspecific binding was assessed in the presence of 4 μM (+)-butaclamol. Bound ligand was separated from free by filtration through a PerkinElmer Unifilter-96 GF/C 96-well microplate using the PerkinElmer Unifilter-96 Harvester, followed by washing three times, 1 mL per well, with ice-cold assay buffer. After samples had dried, 50 μL of liquid scintillation cocktail (MicroScint PS, PerkinElmer, Waltham, MA) was added to each well and plates were sealed and analyzed on a PerkinElmer Topcount NXT instrument.

PMN-MDSCs (Ly6G⁺) were purified from spleens and tumors. Isolated cells were subsequently incubated with biotinylated Ly6G antibody and streptavidin microbeads (Miltenyi). M-MDSCs (CD45⁺CD11b⁺Ly6C^hiLy6G⁻) and macrophage (CD45⁺CD11b⁺F4/80⁺Ly6C⁻) were sorted using FACS Aria (BD Biosciences). PMN-MDSC, M-MDSC or macrophages were plated in U-bottom 96-well plates (3 replicates) in RPMI supplemented with 10% FBS, Penicillin-Streptomycin and 0.05 mM 2-mercaptoethanol, and co-cultured at different ratios with splenocytes from PMEL mice in the presence of 0.1 μg/mL of murine gp100 peptide (EGSRNQDWL, AnaSpec, Inc.). After 48 h, the cells were incubated with ³H-thymidine (1 μCi/well; GE healthcare) for 16 h. Proliferation was measured using the TopCount NXT instrument (PerkinElmer).

Single cells suspensions from spleen and tumors were prepared as described above. Then cells were stained and sorted on BD FACS Aria BD (Biosciences). PMN-MDSC (CD45⁺CD11b⁺Ly6G⁺Ly6C^lo) and M-MDSC (CD45⁺ CD11b⁺Ly6G⁻Ly6C^hi) were plated in U-bottom 96-well plates (3 replicates) in RPMI with 10% FBS and co-cultured at different ratios with splenocytes from Pmel or OT-1 transgenic mice in the presence of cognate peptides: OT-1 (SIINFEKL; 0.1 ng/ml), Pmel (EGSRNQDWL; 0.1 μg/ml). After 48 h, cells were incubated with [³H]-thymidine (PerkinElmer) for 16–18 h. Proliferation was measured by using TopCount NXT instrument (PerkinElmer).

BM Ly6G⁺ neutrophils were prepared as described in Isolation of mouse neutrophils. Then cells were plated in U-bottomed 96-well plates in triplicates in complete RPMI and co-cultured at various ratios with total splenocytes from Pmel or OT-I mice in the presence of cognate peptides (OT-I, SIINFEKL, Anaspec Cat#AS-60193-1; Pmel, EGSRNQDWL, Cat# Anaspec AS-64752) for 48h. Then [³H] thymidine (PerkinElmer, Cat#NET027X001MC) was added (1μl/well), followed by incubation overnight. Proliferation was measured with a TopCount NXT instrument (PerkinElmer).