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The base medium for this cell line is MCDB-131 (Gibco cat # 10372-019). To make the complete medium add to 500 mL of the base medium:

• 56 mL Fetal Bovine Serum (FBS; ATCC 30-2020)

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with DPBS (ATCC catalog # 30-2200) to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
   Cultures can be established between 2.0 x 10⁴ and 4.0 x 10⁴ viable cells/cm².
6. Incubate cultures at 37°C.

Candal FJ, et al. BMEC-1: a human bone marrow microvascular endothelial cell line with primary cell characteristics. Microvasc Res 52(3):221-34, 1996. PubMed: 8954864