Isolation and Activation of Naive B Cells

Naïve B cells from spleens of VI10YEN and KL25 mice were negatively selected by magnetic isolation with CD43 beads (Miltenyi Biotec), as described (37 (link)). The purity was ~98% as determined by B220 staining. Purified B cells (10⁶ cells / ml) were cultured in the presence of PFA-inactivated VSV or LCMV (0.5 m.o.i. equivalent). In some experiments, B cells were harvested after 24 hours and analyzed by flow cytometry for the surface expression of activation markers (see below). In other experiments, B cells were cultured for 72 hours and the supernatants were tested for the presence of total mouse IgM by ELISA.

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Sammicheli S., Kuka M., Di Lucia P., de Oya N.J., De Giovanni M., Fioravanti J., Cristofani C., Maganuco C.G., Fallet B., Ganzer L., Sironi L., Mainetti M., Ostuni R., Larimore K., Greenberg P.D., de la Torre J.C., Guidotti L.G, & Iannacone M. (2016). Inflammatory monocytes hinder antiviral B cell responses. Science immunology, 1(4), eaah6789.

Publication 2016

CD43 beads

B cells Cd43 Cells Elisa Flow cytometry Isolation Lcmv Mouse

Corresponding Organization : Istituti di Ricovero e Cura a Carattere Scientifico

Other organizations : University of Basel, The San Raffaele Telethon Institute for Gene Therapy, Fred Hutch Cancer Center, Scripps Research Institute

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Variable analysis

independent variables

Mice strain (VI10YEN and KL25)

dependent variables

Purity of B cells (as determined by B220 staining)
Surface expression of activation markers (on B cells after 24 hours of culture)
Total mouse IgM in B cell culture supernatants (after 72 hours of culture)

control variables

Method of B cell isolation (negatively selected by magnetic isolation with CD43 beads)
B cell culture conditions (10^6 cells/ml, with PFA-inactivated VSV or LCMV at 0.5 m.o.i. equivalent)

positive controls

Not specified

negative controls

Not specified

Annotations

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