Macs magnetic separator

Free merozoites were purified as reported^{46 (link)}. Briefly, parasites were synchronized using sorbitol treatment and heparin (Mochida Pharmaceutical Co., Tokyo, Japan). Late-stage parasites (32 to 36 hpi) were purified (>95% purity) by magnetic separation with a MACS magnetic separator (Miltenyi Biotec, Cambridge, MA), incubated with 10 μM E64 (Sigma-Aldrich Corporation, St. Louis, MO) for 6 to 8 h, and pelleted. The schizonts were then resuspended in incomplete culture medium and filtered through a 1.2-μm Acrodisc 32-mm syringe filter (Pall Corporation, Port Washington, NY) to isolate free merozoites.

Adult cortical microglia were isolated using MACS magnetic bead separation (Miltenyi Biotec, San Diego, CA) as described previously [17 (link), 45 (link)]. Briefly, saline-treated and LPS-treated mice were deeply anesthetized and transcardially perfused with 50 mL ice-cold PBS. After removal of the meninges, the cerebral cortices of two mice were harvested and pooled per sample (2 mice = 1 n) and suspended in Hank’s balanced salt solution (HBSS) without CaCl₂ and MgCl₂ (Corning, Corning, NY). A single-cell suspension was prepared using the Miltenyi Neural Tissue Dissociation Kit according to the manufacturer’s instructions. The cells were depleted of myelin by suspension in 3 mL of 30% isotonic Percoll™ (GE Healthcare Life Sciences, Pittsburgh, PA) followed by a 10-min centrifugation at 700 x g at 4 °C. The cell pellet was washed in 5 mL HBSS without CaCl₂ and MgCL₂, and isolation of microglia was performed with magnetic CD11b microbeads (Miltenyi) and MACS magnetic separator (Miltenyi) according to the manufacturer’s instructions.

B16F10 tumors were collected at D12, weighed, and placed in ice cold PBS. Dissociation was performed using the mouse tumor dissociation kit for soft tumors (Miltenyi Biotec). Immune cells were labeled with CD45 MicroBeads (Miltenyi Biotec) and a MACS magnetic separator was used for isolation (Miltenyi Biotec). Isolated CD45⁺ cells were incubated with live/dead fixable dye (Invitrogen; 1:1,000) and Fc block rat anti-mouse CD16/32 (BD biosciences, clone 2.4G2; 1:1,000) for 20 minutes at 4ºC. Following washes with MACS buffer (PBS supplemented with 0.5% BSA, 2 mM EDTA, 2 mM L-Glutamate, 1 mM sodium pyruvate, 0.45% D-glucose, and nonessential and essential amino acids), cells were stained in MACS buffer with brilliant staining buffer plus (1:10, 566385, BD biosciences) and primary antibodies against extracellular markers. For extracellular staining only, cells were fixed with 2% PFA and then washed. For intracellular staining, cells were fixed with FOXP3/transcription factor staining buffer set (Invitrogen) and stained with Foxp3 antibody according to the manufacturer’s protocol. All antibodies are listed in Supplemental Table 4. Cells were assessed on a CytoFLEX LX flow cytometer (Beckman Coulter) and obtained data were analyzed with FlowJo software, version 10.8.1 (FlowJo LLC, BD Biosciences) (see Supplemental Data File 2 for gating strategies).