K2edta coated microtainer tubes

Blood was obtained from mice and immediately added to K₂EDTA-coated Microtainer tubes (BD, Franklin Lakes, NJ) to prevent clotting. The proportions of circulating neutrophils and monocytes within the white blood cell population was determined using the ADVIA® 120 Haematology System (Siemens Healthcare Diagnostics Inc, Tarrytown, NY).

At the time of euthanasia, mice were subjected to isoflurane overdose and blood was collected via cardiac puncture into K2EDTA coated microtainer tubes (BD Pharmingen, cat# 365967). Automated complete blood count with differential was performed using a Hemavet 950FS instrument (Drew Scientific).

Prior to staining, tumors were weighed, mechanically dissociated, incubated in a shaker at 37°C for 30 minutes in RPMI-1640 medium containing 5% fetal bovine serum (FBS), 5 mg/mL collagenases IV and I (Gibco), and 40 U/mL DNase, and then passed through a 70-μm filter as a single-cell suspension. Spleens were crushed through a 70-μm filter and red cell lysis was performed with ammonium chloride–potassium (ACK) buffer (Gibco). Blood was collected by submandibular bleeding with a lancet into K2EDTA coated Microtainer tubes (BD Biosciences) followed by red cell lysis with ACK. Antibodies used for flow cytometry are listed in Supplemental Table 5. Cells were stained for cell surface expression in flat-bottom 96-well plates on ice in phosphate-buffered saline (PBS) with 2% FBS. Intracellular markers were stained using the eBioscience Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer’s instructions. Fluorescently conjugated antibodies were used as per the manufacturers’ instructions. LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used to gate on live cells. Data were acquired on an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed via FlowJo (Becton Dickinson). The gating strategy used for analysis is shown in Supplemental Table 6 and Supplemental Figure 5.