Which cells do you want to use out of the BM population? You can lyse the RBCs, isolate the Low density cells by Percoll gradient and store the cells on ice in the dark overnight. We do not see a significant loss of cells this way.

For best results I strongly suggest you dilute the bone marrow sample 1:2 in PBS and isolate the PBMC by density using Histopaque 1077 or other similar reagent. Take the number of cells required for your experiment and freeze the remaining cells in usable aliquots. My lab uses freezing medium consisting of 40%FBS, 50% RPMI 1640 and 10% DMSO. Slowly freeze the cells at -80oC (-1oC/min) and transfer (24-48hrs) to liquid nitrogen. In our hands we routinely achieve >85% viability.

The reason the isolate the PBMC prior to freezing is three fold. First, you want to eliminate the RBC prior to freezing because they will lyse on thawing and is toxic to the cells. The second reason, the the polymorphonuclear cells (neutrophils, basophils and eosinophils) don't usually freeze well and often lyse during thawing releasing their DNA resulting in significant loss of PBMC. Third, 250 ml of bone marrow is a precious commodity so care should be used to optimize the viability and yield of the cells.

As far as time at room temperature...we always keep our on ice when doing any assay. Room temperature is not a good option because you want to keep any cellular metabolism at a minimum. Just keep the centrifuge tube with your cells in a bucket of ice as you work. I have had them on ice for a 4-5 hour process with excellent viability, even before I started lysing the RBCs.

i want to store bone marrow cells for PCR and Alu sequencing. How long can I keep in 4 degree and what is the long term storage options?