Human B cell isolation and culture
实验概要
This protocol provides a general protocol for human B cell isolation and culture.
实验步骤
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.
-
项目成果
