B cell isolation & stimulation with pluriBead

Isolation

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

Anaylsis

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.

facs analysis b cell isolation
Fig 1: Purity of isolated & stimulated B lymphocytes

elispot analysiselispot analysiselispot analysis
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)

elispot analysiselispot analysiselispot analysis
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.)