D267-3 For Research Use Only. Page 1 of 2 Not for use in diagnostic procedures.

MONOCLONAL ANTIBODY CD267/TACI Code No. Clone Subclass Quantity Concentration D267-3 11H3 Mouse IgG2a  100 g 1 mg/mL

BACKGROUND: CD267, also known as TACI SPECIES CROSS REACTIVITY: (transmembrane activator and calcium modulator and Species Human Mouse Rat cyclophilin ligand interactor) or TNFRSF13B, is a member of the tumor necrosis factor (TNF) receptor family that Cell transfectant Not Tested Not Tested serves as a receptor for B-cell activating factor of the TNF family (BAFF/CD257) and as a proliferation-inducing Reactivity on FCM + ligand (APRIL/CD256). BAFF and APRIL bind both TACI and BCMA (B cell maturation Ag/CD269), with BAFF additionally and exclusively binding the BAFF receptor INTENDED USE: (BAFF-R/CD268) while APRIL can also bind to heparin For Research Use Only. Not for use in diagnostic procedures. sulfate proteoglycans (HSPG). TACI is expressed on B cells and activated T cells. BCMA is exclusively expressed on B cells, whereas BAFF-R is expressed mainly on B cells REFERENCES: but also on T cells. It is reported that mutations of 1) Sakurai, D., et al., Blood 109, 2961-2967 (2007) CD267/TACI are showed in patients with IgA deficiency 2) Hase, H., et al., Blood 103, 2257-2265 (2004) and common variable immunodeficiency (CVID). Sakurai Clone 11H3 is used in reference number 1). et al., reported that TACI positively regulated APRIL-induced IgA production in collaboration with HSPG and TACI negatively regulated BAFF-induced B-cell proliferation and production of IgA and IgG.

SOURCE: This antibody was purified from hybridoma (clone 11H3) supernatant using protein A agarose. This hybridoma was established by fusion of mouse myeloma cell NS-1 with C57BL/6 mouse splenocyte immunized with human TACI full-length cDNA-transfected cells.

FORMULATION: 100 g IgG in 100 L volume of PBS containing 50% glycerol, pH 7.2. No preservative is Flow cytometric analysis of CD267 contained. expression on transfectant. Open histogram indicates the reaction of isotypic control to the cells. Shaded histogram STORAGE: This antibody solution is stable for one year indicates the reaction of D267-3 to the cells. from the date of purchase when stored at -20oC.

REACTIVITY: This antibody reacts with CD267 antigen on Flow cytometry. Clone 11H3 has an agonistic activity and it doesn’t block binding of BAFF to TACI.

APPLICATIONS: Western blotting; Not tested Immunoprecipitation; Not tested Immunohistochemistry; Not tested Immunocytochemistry; Not tested Flow cytometry; 10 g/mL (final concentration) Flow cytometric analysis of CD267 Detailed procedure is provided in the following expression on EBV-LCL cells. Shaded PROTOCOL. histogram indicates the reaction of isotypic control to the cells. Open histogram indicates the reaction of D267-3 to the cells.

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL https://ruo.mbl.co.jp e-mail [email protected], TEL 052-238-1904 D267-3 Page 2 of 2

PROTOCOL: D222-4 FITC labeled anti-mouse GITR (DTA-1) Flow cytometric analysis for floating cells D222-5 PE labeled anti-mouse GITR (DTA-1) We usually use Fisher tubes or equivalents as reaction tubes K0031-3 anti-HVEM (122) for all steps described below. K0031-4 FITC labeled anti-HVEM (122) 1) Wash the cells 3 times with washing buffer [PBS MD-10-3 anti-human Fas (CD95) (UB2) MD-10-4 FITC labeled anti-human Fas (CD95) (UB2) containing 2% fetal calf serum (FCS) and 0.1% NaN3]. 2) Resuspend the cells with washing buffer (5 x 106 MD-10-5 PE labeled anti-human Fas (CD95) (UB2) cells/mL). MD-11-3 anti-human Fas (CD95) (ZB4) 3) Add 50 L of the cell suspension into each tube, and SY-001 anti-human Fas (CD95) (CH-11) centrifuge at 500 x g for 1 minute at room temperature D041-3 anti-human FasL (CD178) (4H9) (20~25oC). Remove supernatant by careful aspiration. D041-4 FITC labeled anti-human FasL (CD178) (4H9) D041-5 PE labeled anti-human FasL (CD178) (4H9) 4) Add 20 L of Clear Back (human Fc receptor blocking reagent, MBL; code no. MTG-001) to the cell pellet after D041-6 Biotin labeled anti-human FasL (CD178) (4H9) tapping. Mix well and incubate for 5 minutes at room D042-3 anti-human FasL (CD178) (4A5) temperature. D026-3 anti-mouse Fas (CD95) (RMF2) D027-3 anti-mouse Fas (CD95) (RMF6) 5) Add 40 L of the primary antibody at the concentration as D057-3 anti-mouse FasL (CD178) (FLIM58) suggest in the APPLICATIONS diluted in the washing D057-4 FITC labeled anti-mouse FasL (CD178) (FLIM58) buffer. Mix well and incubate for 30 minutes at room D057-6 Biotin labeled anti-mouse FasL (CD178) (FLIM58) temperature. D069-3 anti-mouse FasL (CD178) (FLIM4) 6) Add 1 mL of the washing buffer followed by M031-3 anti-TRADD (3E11) centrifugation at 500 x g for 1 minute at room temperature. K0033-3 anti-DR3 (B65) Remove supernatant by careful aspiration. K0033-4 FITC labeled anti-DR3 (B65) 7) Add 30 L of 1:100 FITC conjugated anti-mouse IgG M112-3 anti-mouse TRAF2 (6F8) (MBL; code no. 238) diluted with the washing buffer. Mix 592 anti-mouse TRAF2 (polyclonal) well and incubate for 30 minutes at room temperature. 597 anti-mouse TRAF6 (polyclonal) 8) Add 1 mL of the washing buffer followed by M092-3 anti-TRAF6 (1F8) centrifugation at 500 x g for 1 minute at room temperature.

Remove supernatant by careful aspiration.

9) Resuspend the cells with 500 L of the washing buffer

and analyze by a flow cytometer.

(Positive controls for Flow cytometry; transfectant,

EBV-LCL)

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