Hi Rohan!

I believe you're talking about yellowish clouds at the centre of the well or flasks.

Usually, mononuclear cells from blood and BM tend to collect at the centre of culture vessels, mainly if they were isolated with hard-hitting reactives, like red blood lysis buffer.

What I would recommend for you is to look at these clouds under the light microscope to be sure that you are dealing with the cells. Also, be sure that your cell density is optimal for your culture vessel as it may be too high.

By the way, I would also try to analyse cell viability after this isolation method as red blood lysis is generally not recommended for cells that are to be cultured. They become quite lazy to differentiate into anything and tend to die quickly.

I hope this will help you to culture these amazing cells.

Hi Rohan Bir Singh . Hopefully it is not contamination. Apart from cell debris as Kirill mentioned above, it is also likely that you have megakaryocytes in your bone marrow prep. They can bud off platelets that make the media look cloudy.

Hi Rohan!

Bone marrow is rich in preadipocytes, adipocytes and also acellular lipide droplets of different size. These elements are floating and even careful washes very difficult to remove. If you has these they should be carefully removed from the surface layer preferably under a stereomicrocope.

In addition whole bone marrow aspirates always contain the blood forming hematon units.

You may find help in our technical protocol paper under the above adress; and in our publications.

Good luck

Istvàn

ProtocolExchange/DOI:10.1038/protex.2013.006 ;or

http://www.nature.com/protocolexchange/protocols/2553

Last time I cultured bone marrow cells in rpmi it took me like 15 attempts to perfect it. Problem with primary cell culture is contamination from yeast cells...it gets cloudy overtime and you can see those large cells without microscope I even had to face a film floating over media.

There are several basic astuces you should check before throw-away your dish; I mention here just some possibilities which "normally" complicate the initial step of preparation.

1) Origin of bone marrow sample - human or mice?

Human BM is rich in floating, yellowish, sticky lipid, rich in retinoids. They tend to appear on the top layer as floating refractory droplets preferentially at the center of dish.

2) Due to tensil forces the floating material may slide to the centrifugal direction, and may stick to the vertical plastic surface that you even "miss" to see!

3) You remooved RBCs by hypotonic lysis.

Are You sure that the residual RBC membrane material was eliminated by the subsequent washes?

4) Contamination?

As contamination is easy to get, use sterile environment, instruments and AB/AMy containing medium during the initial preparations; a so rapid (3d) massive contamination occurs rarely; and surely not systematically!

5) Un easy way to identify the material in question is to take carefully 100-200 µL from the claudy material and analyse the content under a phase contrast microscope at low and at high magnification!

6) Finally, in a high cell density culture you may find many nonadherent cells, and among, you may have large nodular spheroids, that we have identified some 30 years ago and named "the haematon". Since it appeared that they represent blood forming tissu units 50 -to 200 µm in diameter.

If it is so, You may find a lot of practical aspects about the haematons in our publication and you can see these at

nature.com or ProtocolExchange/DOI:10.1038/protex.2013.006

http://www.nature.com/protocolexchange/protocols/2553

Protocol

Purification and processing of blood-forming tissue units,

the haematons, in searching for mammalian stem cell niches

Blazsek I et al

If you could show me the image I could help you better