B cell isolation & stimulation with pluriBead
|Sample Material||50ml EDTA whole blood|
|Isolation method||Non-magnetic with 300 µl CD19 S-pluriBeads® anti-human|
|Yield||~3.5 * 10^6 B lymphocytes|
|Vitality||>99% (Trypan blue staining)|
|Purity determination||Anti-CD20-FITC antibody staining followed by FACS analysis|
|Purity||~97% B lymhocytes|
Frequency determination of antigen specific Memory B cells
After target activation, frequency of antibody secreting plasma cells (ASC) was determined via ELISpot.
Isolated B cells had been stimulated for 5 days with a polyclonal activation mixture. Afterwards, they had been grown on a plate coated with the specific antigen. After 24h incubation, the assay had been processed.
Spots had been counted and evaluated with a specific Software (AID ELISpot6.0 iSpot). In the adjacent diagram, the blue numbers reflect the count of antigen-specific spots per well.
Fig 1: Purity of isolated & stimulated B lymphocytes
Fig 2: ELISpot analysis of anti-Ig specific cells in duplicates. d1d2: 250 cells/well each, d3d4: 500 cells/well each, d5d6: non-activated cells (500 cells/ well each)
Fig 3: d7d8: anti-influenza-nucleocapsid specific Memory B cells, d9d10: nti-Tetanus-toxoid specific Memory B cells, d11d12: Negative control
Interpretation / Summary
S-pluriBead® CD19 allowed for isolating B lymphocytes from a human whole blood sample with a purity of ~ 97%.
The isolated targets showed a vitality of >99 % after Trypan blue staining. They had been successfully grown in cell culture as well as differenciated to antibody secreting plasma cells (ASC). ASCs proved suitable for analysis of antigen-specific memory B-Cell response via ELISpot assay. Results had been comparable to those gained from a reference method (Dynabeads® CD19 Pan B).
For reference method see Publication: Bussmann BM, Reiche S, Bieniek B, Krznaric I, Ackermann F, Jassoy C: Loss of HIV-specific memory B-cells as a potential mechanism for the dysfunction of he humoral immune response against HIV. Virology 2010; 397(1):7-13.)