Antibodies binding to CD34+/CD36+ fetal but not to adult cells
A fetal cell, cell antigen technology, applied in the field of antibodies that bind to fetal but not adult CD34+/CD36+ cells
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example 1
[0044] Example 1: Preparation of fetal liver cells
[0045] Fetal liver cells can be obtained from Cambrex Bio Science, Walkersville, M.D. Alternatively, it can be isolated from fetal livers (FL, 15-22 weeks of gestation) from 5 different donors, as described below.
[0046] Fetal livers from five donors were homogenized in DPBS / 0.2% BSA solution and passed through a metal sieve. Among them, DPBS does not contain Ca ++ / Mg ++ Dulbecco's phosphate buffered saline solution (Biowhittaker, Walkersville, MD); BSA is bovine serum albumin (Sigma St. Louis, MO), and contains 50 μg / ml gentamicin sulfate (Life Technologies, Grand Island NY). CD34 + Cells can be sorted from the homogenate using MACS or FACS as described in Example 3. Or, in accordance with the MACS or FACS method described in Example 3 to sort CD34 + Before the cells, the homogenate was further purified as described below.
[0047] Use immunomagnetic microspheres to exclude mature red blood cells and other lineage-positive c...
example 2
[0048] Example 2: Preparation of mobilized peripheral blood cells
[0049] The mobilized peripheral blood cells are obtained from Cambrex, and can also be obtained from the following methods.
example 3
[0052] Example 3: Adult and fetal CD34 + Purification of progenitor cells
[0053] The fetal liver cells and MPB mononuclear cell fractions of Example 1 and Example 2 were recorded in CD34 according to any of the following methods. + Cell enrichment.
[0054] MACS method
[0055] Fetal liver cells and MPB monocytes were prepared according to the methods of Examples 1 and 2, respectively, and MACS CD34 was used + The separation kit (Miltenyi Biotec, Auburn, CA) is in accordance with the instructions for use in CD34 + Cellular immune magnetic beads are enriched. Briefly, monocytes are incubated with hapen-labeled anti-CD34 antibody (QBEND-10, BD Pharmingen, San Diego, CA) in 0.1% human IgG (Bayer Elkhart, IN) as a blocking agent, and then ligated with Co-culture to MACS microbeads against hapen. The labeled cells are filtered through a 30μm nylon mesh to remove cell clumps and aggregates. Then labeled CD34 + The cells are captured from the mixture through a high gradient magnetic se...
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