Anti cd71 fitc

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).

ENU (CAS no. 759–73-9) and EMS (CAS no. 62–50-0), heparin sodium, low melting point agarose, Triton X-100, dimethyl sulfoxide were purchased from Sigma-Aldrich (Shanghai, China). Anti-CD59-APC and anti-CD71-FITC were obtained from BD Biosciences (San Jose, CA, USA). Anti-CD61-PE and anti-CD45-PE were purchased from eBiosciences (San Diego, CA, USA). DRAQ5 was provided by Abcam (Cambridge, UK). SYTO13 and propidium iodide (PI) were supplied by Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Phosphate-buffered saline (PBS) and Hanks balanced salt mixture (HBSS) and fetal bovine serum (FBS) were purchased from Gibco, Thermo Fisher Scientific (Waltham, MA, USA). Normal melting point agarose and ethylenediaminetetraacetic acid (EDTA) and Tris base were purchased from Amresco (Shanghai, China).

Briefly, for the cellular differentiation assay, the cells were seeded at a concentration of 1 × 10⁵ cells/well in 6-well culture plates and incubated with increasing doses of BW18 (5, 10 and 20 µM) for 48 h and 72 h. Cells were washed and resuspended with 100 µL ice-cold PBS. Anti-CD41-FITC, Anti-CD61-APC, Anti-CD71-FITC and Anti-CD235a-APC (BD Biosciences) were, respectively, added to the cell suspension. After 30 min of incubation on ice, cells were washed and resuspended in 500 μL PBS. Quantification of positive cells was analyzed with FACS Calibur flow cytometer (BD Biosciences).