Spleens and draining lymph nodes (DLNs) from 4T1 and 4T1.GCSF− tumor-bearing mice were isolated when tumors reached 500–700 mm3. Single cell suspensions were prepared by mechanically dissociating spleen and DLNs with a syringe plunger and passing samples through a 40-μm nylon mesh cell strainer. Splenocytes were additionally treated with ammonium-chloride-potassium buffer (Lonza, Allendale, NJ, USA) for 10 min to lyse red blood cells. Single cell suspensions were then blocked with purified rat anti-mouse CD16/CD32 monoclonal antibody (BD Biosciences) and stained with fluorochrome-conjugated anti-CD11b (clone M1/70), anti-CD19 (clone 1D3), anti-Ly6G and Ly6C (clone RB6-8C5), anti-CD25 (clone PC61), anti-CD4 (clone GK1.5), and anti-CD3ε (clone 145-2C11) (BD Biosciences).
Cells were then rinsed, fixed and permeabilized with 1× Perm/Wash buffer from BD Biosciences. The permeabilized cells were further stained with fluorochrome-conjugated anti-FoxP3 and read on a BD FACSCanto II flow cytometer. Frequencies of MDSCs, T cells, B cells, and regulatory T cells (Tregs) in the single cell suspensions were determined using FlowJo software (Tree Star, San Carlos, CA, USA). For mice bearing 4T07, 67NR, 66Cl4 and 168FARN tumors, only the spleens were isolated and stained for MDSCs.
Cells were then rinsed, fixed and permeabilized with 1× Perm/Wash buffer from BD Biosciences. The permeabilized cells were further stained with fluorochrome-conjugated anti-FoxP3 and read on a BD FACSCanto II flow cytometer. Frequencies of MDSCs, T cells, B cells, and regulatory T cells (Tregs) in the single cell suspensions were determined using FlowJo software (Tree Star, San Carlos, CA, USA). For mice bearing 4T07, 67NR, 66Cl4 and 168FARN tumors, only the spleens were isolated and stained for MDSCs.