A new method for detachment of Dynabeads from positively selected B lymphocytes.
This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.
Laboratory of Immunology, Institute for Cancer Research, Montebello, Oslo, Norway.
A M
AM
Rasmussen
E B
EB
Smeland
B K
BK
Erikstein
L
L
Caignault
S
S
Funderud
D015703:Antigens, CD / Q000032:analysis*; D018951:Antigens, CD20; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D002448:Cell Adhesion; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D002472:Cell Transformation, Viral; D004854:Herpesvirus 4, Human / Q000254:growth & development; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D066298:In Vitro Techniques; D037181:Lectins, C-Type; D008280:Magnetics; D011951:Receptors, Complement / Q000032:analysis; D017464:Receptors, Complement 3d; D011961:Receptors, Fc / Q000032:analysis; D017455:Receptors, IgE; D000099192:CD69 Antigens
D015703:Antigens, CD; D018951:Antigens, CD20; D000944:Antigens, Differentiation, B-Lymphocyte; D000945:Antigens, Differentiation, T-Lymphocyte; D007140:Immunoglobulin Fab Fragments; D037181:Lectins, C-Type; D011951:Receptors, Complement; D017464:Receptors, Complement 3d; D011961:Receptors, Fc; D017455:Receptors, IgE; D000099192:CD69 Antigens