CD273 (B7-DC) Monoclonal Antibody (TY25), PE, eBioscience™

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mice were sacrificed and spleens were used for preparing single-cell suspensions by mechanical dissociation followed by removal of red blood cells with ammonium chloride lysis buffer (ACK).

Upstream tips
mice were sacrificed and spleens were used for preparing single-cell suspensions by mechanical dissociation followed by removal of red blood cells with ammonium chloride lysis buffer (ACK).

Publication protocol

MDSCs were cultured in RPMI medium containing 10 ng/ml GM-CSF under hypoxia (0.1% pO2 with 5% CO2) in a hypoxia chamber (InVivo2 400 Hypoxia Workstation; Ruskinn). For CoCl2 experiments, B16-F10 tumor-bearing C57BL/6 mice or 4T1 tumor-bearing BALB/c mice were injected i.p. with PBS1X and either 30 or 60 mg/kg of CoCl2 (200 μl per mice). After 6 h, mice were sacrificed and spleens were used for preparing single-cell suspensions by mechanical dissociation followed by removal of red blood cells with ammonium chloride lysis buffer (ACK). Total splenocytes were immediately stained for MDSCs (Gr1+ CD11b+), macrophages (F4/80+), and DCs (CD11c+) along with either PD-L1-PE or PD-L2-FITC conjugated antibodies and analyzed by flow cytometry.

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