HumanBcellisolationandculture

實驗概要

This protocol provides a general protocol for human B cell isolation and culture.

實驗步驟

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.