Homogenizing femurs and tibias to obtain bone marrow cells.

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Put the 2 femurs and 2 tibias dissected from one mouse into the mortar, and add 5 ml of cold, sterile MACS buffer. Firmly hold the pestle and apply downward force to grind up the femurs and tibias. After the femurs and tibias are broken into pieces, use circular motion to fully release the bone marrow cells within the bone cavities. Apply circular grinding motion for 25 times clockwise and 25 times counter clockwise. For the isolation of BMECs as we discussed in this protocol, both the white bony tissues and the cell suspension are collected into the 15 ml Falcon tube, followed by enzymatic digestion. For collecting hematopoietic cells to retrieve Lin- cells for coculture, collect 5 ml of supernatant containing the cell suspension and immediately filter through a 40 μm cell strainer into a 50 ml Falcon tube. Add 5 ml MACS buffer into the white bony remains and apply circular grinding motion. Collect the supernatant until the red bone marrows have been completely rinsed out, leaving only white bone tissues. Usually, it takes 3 rounds of 5 ml MACS buffer and circular grinding to complete the homogenization processes. No enzymatic digestion (collagenase and dispase) is needed. In total, for collecting the hematopoietic stem cells, we have about 15 ml cell suspension in MACS buffer, ready for downstream processing.

Extracted from protocol
Generation of BMEC Lines and in vitro BMEC-HSPC Co-culture Assays
Guo, P. and Rafii, S. (2018). Bio-protocol 8(21): e3079.
DOI: 10.21769/BioProtoc.3079.