Anti ter119 apc

E9.5 and E10.5 whole embryos or dissected yolk sacs were disaggregated with 0.25% collagenase type I (Stemcell Technologies) at 37°C for 30 min, and the cells were washed with PBS containing 2% FBS (Gibco) and filtered through a 70‐μm mesh. The single‐cell suspension was then incubated for 30 min at 4°C with the following antibodies: anti‐CD71‐FITC (BD Biosciences), anti‐Ter119‐APC (BD Biosciences), anti‐cKit‐PEcy7 (BD Biosciences), and anti‐CD41‐PE (BD Biosciences). Samples were analyzed with the BD LSRFortessa flow cytometer.
Bone marrow of adult mice was obtained from femurs and tibias crushed in a mortar and filtered through a 70‐μm mesh to obtain single‐cell suspensions. For hematopoietic cell maturation assays, a small fraction of the bone marrow was separated and the rest was depleted of red blood cells by lysis in FACSLysing solution (BD Biosciences). Antibodies used for blood maturation assay were anti‐CD71‐FITC (BD Biosciences) and anti‐Ter119‐APC (BD Biosciences). Antibodies for BM precursor sorting were Biotinylated lineage cocktail (BD Biosciences), anti‐CD34(RAM34)‐FITC (BD Biosciences), anti‐cKit‐PEcy7 (BD Biosciences), anti‐CD16/32‐BV605 (BD Biosciences), and anti‐Sca1‐PerCP‐Cy5.5 (BD Biosciences).

Single CD34^-LKS cells from C57BL/6 mice were individually deposited into each well of a 96-well plate along with 100 μL of serum free medium (Gibco) containing 100 ng/mL of mouse SCF, 10 ng/mL of mouse IL-3, 25 ng/mL of human TPO, 2 U/mL of human EPO and p18SMI compounds (different dilutions). Cells were cultured in a 37 °C, 5% CO₂ incubator for 14 days for ex vivo colony formation. Colonies were then cytospined and analyzed using flow cytometry. Neutrophils (n), macrophages (m), erythroblasts (E) or megakaryocytes (M) were identified based on their cell surface maker, Gr-1, Mac-1, Ter119 and CD41. Cells were measured on an FACS analyzer (FACSArray, BD Biosciences). Antibodies were used as following: anti-Gr-1-Percp-cy5.5, anti-Mac-1-APC-cy7, anti-Ter119-APC, anti-CD41-PE (BD Biosciences). The selection of cell surface marker of each subpopulation cells were considered based on the manufacture’s guide (BD Biosciences). To be specific, Gr-1 cell marker represents neutrophils (n), Mac-1 represents macrophages (m), Ter119 represents erythroblasts (E) and CD41 represents megakaryocytes (M). The wells from each group that generated all lineages (nmEM) were counted. The ratio of p18SMI compound treatment group to the control group (DMSO-treated) was calculated and fitted to a curve to calculate the ED₅₀ value using GraphPad Prism v6.0.