PMC:2195754 (5) JSONTXT < >
|Materials and Methods|
Analysis of Normal B Cell Populations.
Tonsillar mononuclear cells were sequentially incubated with CD27-FITC (PharMingen) and anti-FITC microbeads (Miltenyi Biotec), then passed over a CS-MACS column (Miltenyi Biotec). CD27− cells were sequentially incubated with mouse anti–human IgD (Southern Biotechnology Associates) and anti–mouse IgG2a/b microbeads (Miltenyi Biotec), and IgD+ cells were isolated via an LS-MACS column (Miltenyi Biotec). Tonsillar mononuclear cells from the same patient were incubated consecutively with rat anti–human CD77 (Immunotech), mouse anti–rat IgM (Serotec), and anti–mouse IgG1 microbeads (Miltenyi Biotec), and CD77+ cells were isolated via an LS-MACS column. Total genomic DNA was isolated from both cell populations, and exons 1 and 2 of IκBα were amplified as above using PfuTurbo™ polymerase (Stratagene). After 35 cycles, PCR products were purified, incubated with 200 μM dNTPs, and Taq polymerase (GIBCO BRL) for 15 min at 72°C, and cloned into the pGEM-T Easy vector (Promega).
|Unselected / annnotation||Selected / annnotation|