Year: 2007
Type: article
Abstract: 1. Four B6 mice are sacrificed and dissected to obtain tibias, femurs, hip and spine.2. During the dissections, the limbs and the bones are kept in PBS 2%FCS heat inactivated (optional 2mM EDA -dissection medium).3. The clean bones are crushed with mortar and pestle in dissection medium.An efficient way to do it is to crush tibias, femurs and hips of each mouse, then 2 spines at a time.4. The cell mixture obtained from each mouse is kept separate and filtered through a 40µm filter into a 50ml falcon tube.In this way, each tube contains the cell mixture obtained out of all the leg bones of a mouse, and half of the mixture obtained from 2 spines.At this stage, the filter is used to separate bone debris.5.It is usually advisable to crush the leg bones twice to obtain white (with no bone marrow) bone fragments, and the spine fragments 2 or 3 times.6. Fill up the tubes to have them balanced, and spin 5 min 1200rpm.7. Aspirate the supernatant and loosen the pellet by dragging the tubes on the tube rack (this step is important after each centrifugation to minimize clump formation and cell loss).Resuspend each tube in 1 ml PBS 2% FCS (NO EDTA from now on) if you are doing lineage depletion, or in whatever volume is otherwise convenient for you.
DOI: 10.3791/157
Authors: Cristina Lo Celso, David Scadden
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2052214227