Anti gr 1 rb6 8c5

RPMI1640, DMEM, fetal bovine serum (FBS) and antibiotics were obtained from HyClone. Recombinant murine GM-CSF, IL-6, TNFα and IL-4 were purchased from Peprotech. PGE2 and 2-ME (2-Mercaptoethanol) were from Sigma. The following Abs were used, including PE-, or FITC- conjugated anti-Gr-1 (RB6-8C5), anti-CD11b (M1/70), anti-Ly6G (1A8), anti-Ly6C (AL21), anti-CD80 (16-10A1), anti-CD86 (GL1), anti- CD40 (3/23), anti-CD11c (N418), anti- PD-1 (J43), anti- B7-H1 (MIH1), anti-CD4 (L3T4), anti-CD8a (Ly-2) and isotype control Abs, which were obtained from BD Biosciences. Purified anti-GM-CSF Ab (MP1-22E9) was purchased from Biolegend.

Implantation sites were harvested as described above. Each site was cut into 12 pieces and placed in RPMI 5% fetal bovine serum (FBS). These pieces were digested in RPMI containing 5% FBS, 300 μg/mL collagenase F (Sigma‐Aldrich), 200 μg/mL collagenase L (Sigma‐Aldrich), 500 μg/mL Dispase (Gibco), and 2 U/mL DNase‐1 (Roche) at 37°C for 30‐45 minutes with a magnetic stir bar for agitation. Cells were passed over a 70 μm strainer (BD) to create a single‐cell suspension. Implantation sites were washed in DPBS, 1% FBS, 25 mmol/L EDTA. Cells were stained for FACS with anti‐CD45 (30‐F11, BD), anti‐CD11b (M1/70, BD), anti‐Gr‐1 (RB6‐8C5, BD), and rabbit anti‐Crry followed by a donkey anti‐rabbit DyLight 488 (Jackson ImmunoResearch). Blocking for FACS was carried out employing DPBS, 1% FBS, 25 mmol/L EDTA with 5% donkey serum, and 5% mouse serum. Cells were examined employing a FACScan (BD) retrofitted with a Cytek Upgrade.