A bit of help from the experts. We had a fresh human bone marrow sample (in EDTA), delivered to us, which we diluted 1:3 in (1x) PBS/2mM EDTA prior to loading it onto Histopaque 1077, centrifuging it for 30' at 400g @ RT.

This is what we got from the bone marrow sample (see pics attached). We also run a peripheral blood sample (freshly taken from the same patient) in parallel, where the PBMC layer came out as expected.

Subsequent centrifugations did indicate a pellet on both samples but then, when the red blood cell lysis buffer was applied (and inactivated with FBS after 8' at 4C), PBMCs on the Bone Marrow sample (but not on the peripheral blood sample) were lysed.

Can someone suggest what might went wrong?

Thank you in advance