Product Protocols: Flow Cytometry and Cytology and and Cell Culture Protocol with BAFF protein (Catalog Number: OPPA00572)

Product Protocols:
Flow Cytometry and Cytology and and Cell Culture Protocol with Gene Name: BAFF Antibody (Catalog Number: OPPA00572)

Catalog Number: OPPA00572

Reference:
1: Chihara N, Aranami T, Sato W, Miyazaki Y, Miyake S, Okamoto T, Ogawa M, Toda T, Yamamura T.Interleukin 6 signaling promotes anti-aquaporin 4 autoantibody production from plasmablasts in neuromyelitis optica.Proc Natl Acad Sci U S A.2011 Mar 1;108(9):3701-6.Epub 2011 Feb 14.PubMed PMID: 21321193; PubMed Central PMCID: PMC3048150.

Product Name: BAFF Human – Recombinant Human BAFF (BLyS) (5ug)

Gene Name: BAFF

Sample description: Binds to tnfrsf13b/taci and tnfrsf17/bcma.tnfsf13/april binds to the same 2 receptors, together, they form a 2 ligands -2 receptors pathway involved in the stimulation of b- and t-cell function and the regulation of humoral immunity.a third b-cell specific baff-receptor (baffr/br3) promotes the survival of mature b-cells and the b-cell response.
B Lymphocyte Stimulator functions as a potent B-cell growth factor in costimulation assays.Administration of BAFF Human recombinant to mice disrupts splenic B-cell and T-cell zones and results in elevated levels of serum immunoglobulin.

Experiment Name: Flow Cytometry and Cytology and and Cell Culture

Experiment Background:
PBMC were separated using density centrifugation on Ficoll-Paque PLUS (GE Healthcare Biosciences).B cells were analyzed and sorted by FACSAria (BD Biosciences).Each B-cell subset was stained with May-Grünwald-Giemsa.To evaluate AQP4-Ab production in vitro, each B-cell subset (1 or 2 × 104) was cultured for 6 d in the medium alone, in the presence of IL-6 (1 ng/mL) or in the presence of IL-6 (1 ng/mL), IL-21 (50 ng/mL), and anti-CD40 (1 ug/mL).Culture supernatants were harvested and analyzed for AQP4-Ab production.To examine the effect of cytokines on the survival of PB, the cells (4 × 103) were cultured in the medium alone or in the presence of BAFF (100 ng/mL), APRIL (300 ng/mL), or IL-6 (1 ng/mL) in 96-well U-bottom plates for 2 d and stained with PI to assess cell survival.In parallel, the cells were cultured for 1 d and harvested to evaluate mRNA expression by qRT-PCR.To assess the effect of IL-6 signaling blockade, PBMC (5 × 105) were preincubated with anti-IL-6R Abs (1 ug/mL) at 4 C for 20 min, cultured in AIM-V medium
(invitrogen) containing 20% of heat-inactivated serum obtained from each patient in 96-well flat-bottom plates for 2 d, and analyzed by flow cytometry.

Primary antibody dilution: Anti-IL-6R Abs (1 ug/mL)

Experiment step:
(i) Norio et al.evaluated the influence of IL-6, BAFF, and APRIL on the survival of PB after 2 d of in vitro culture.Among the added cytokines, only IL-6 was found to significantly promote the survival of PB.They found that the expression of both unspliced [XBP-1(u)] and spliced [XBP-1(s)] forms of XBP-1 mRNA was augmented in PB by the addition of IL-6.These results suggest that IL-6 promoted the survival of PB and enhanced IgG secretion from PB, leading to an increased production of AQP4-Abs in NMO patients..
(ii) However, in this ex vivo study, BAFF did not promote the survival of PB, indicating that PB were not a target for BAFF.They speculate that BAFF might specifically act on an early process of plasma cell differentiation and does not have an influence on cells like PB that have entered a later stage.

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