The frequencies of IgG and IgM secreting antibody secreting cells (ASC)
were determined using ELISpot PLUS for mouse IgG and IgM kits
(MABTECH AB, Nacka Strand, Sweden) as previously published (32 (link), 46 (link)). Briefly, recipient cells isolated from spleen, graft-draining
mediastinal lymph nodes and BM were cultured for 20 h in RPMI media supplemented
with 5% of Fetal Bovine Serum (FBS) in 96-well plates coated with
anti-mouse IgG Ab. Then, either biotinylated-anti-IgG, biotinylated-anti-IgM or
biotinylated Dd or Db molecules (5µg/ml, provided
by the NIH Tetramer Core Facility at Emory University, Atlanta, GA) was added as
detection reagent for two hours at room temperature. After extensive washes, the
plates were incubated with Streptavidin-Alkaline Phosphatase for one hour at
room temperature followed by BCIP/NBT substrate. The numbers of spots per well
and the cumulative spot size distribution were analyzed using an ImmunoSpot
Series 2 Analyzer (Cellular Technology Ltd., Shaker Heights, OH).
were determined using ELISpot PLUS for mouse IgG and IgM kits
(MABTECH AB, Nacka Strand, Sweden) as previously published (32 (link), 46 (link)). Briefly, recipient cells isolated from spleen, graft-draining
mediastinal lymph nodes and BM were cultured for 20 h in RPMI media supplemented
with 5% of Fetal Bovine Serum (FBS) in 96-well plates coated with
anti-mouse IgG Ab. Then, either biotinylated-anti-IgG, biotinylated-anti-IgM or
biotinylated Dd or Db molecules (5µg/ml, provided
by the NIH Tetramer Core Facility at Emory University, Atlanta, GA) was added as
detection reagent for two hours at room temperature. After extensive washes, the
plates were incubated with Streptavidin-Alkaline Phosphatase for one hour at
room temperature followed by BCIP/NBT substrate. The numbers of spots per well
and the cumulative spot size distribution were analyzed using an ImmunoSpot
Series 2 Analyzer (Cellular Technology Ltd., Shaker Heights, OH).
