B cell isolation
1. Donor blood was obtained with informed consent under guidelines issued by the Medical Ethics Committee.
2. Mononuclear cell fractions were isolated by Ficoll-amidotrizoate centrifugation.
3. B
cells were immunomagnetically isolated by positive selection and the
CD19 positively selected cells were released from the beads using
Detach-a-Bead.
4. Alternatively, B cells were isolated with a Dynal kit based on depletion of non-B cells (negative selection).
5. Yields
of positive and negative selections were 98.5 ± 0.5% and 93.8 ± 1.3%
pure B cells, respectively, as assessed by staining with a FITC-labeled
anti CD19 mAb and flow cytometric analysis of lymphocyte gated cells on a
FACSCalibur equipped with CellQuest software.
B cell culture
1. B cells were isolated by the positive or
negative selection methods as described above and were incubated with
tetrameric HLA-A2/HPV (100 ng/106 cells) on ice for 40 min, washed, and
aseptically sorted for PE staining in a FACSVantage SE (BD Biosciences).
2. Cells with PE staining intensity exceeding channel number 10
were deflected and seeded directly at 1/well in 96-well plates that had
been preseeded with EL4.B5 cells (50 Gy irradiated, 5 x 104/well and a T cell supernatant.
3. This
T cell supernatant was produced by culturing E-rosette-enriched T cells
from a male donor for 36 h in the presence of 5 μg/ml PHA and 10 ng/ml
PMA.
4. B cell supernatants (100 μl) were collected at days 9 and 16.
5. All supernatants were analyzed for anti-HLA Ab by CDC and some supernatants for IgG and IgM by ELISA.
6. Some supernatants were tested for binding to streptavidin.
Reference
1. Wen, L., M. Hanvanich, C. Werner-Favre, N. Brouwers,
L. H. Perrin, R. H. Zubler. Limiting dilution assay for human B cells
based on their activation by mutant EL4 thymoma cells: total and
antimalaria responder B cell frequencies. Eur J Immunol. 1987; 17: 887
2. Arend Mulder, Chantal Eijsink, Marrie J. Kardol, Marry E. I. Franke-van Dijk, Sjoerd H. van der Burg, Michel Kester, Ilias I. N. Doxiadis, and Frans H. J. Claas. Identification, Isolation, and Culture of HLA-A2-Specific B Lymphocytes Using MHC Class I Tetramers. J Immunol. 2003; 171: 6599.