D051-4 For Research Use Only. Page 1 of 1 Not for use in diagnostic procedures.

MONOCLONAL FITC labeled CD154 (CD40-Ligand) Code No. Clone Subclass Quantity D051-4 5F3 Mouse IgG1 50 tests

BACKGROUND: The human CD154 (CD40-ligand) is SPECIES CROSS REACTIVITY: a 39 kDa type II membrane glycoprotein predominantly Species Human Mouse Rat expressed on CD4+ T cells, but also on a small proportion of CD8+ cells and NK cells. It is belonging to the tumor Cell transfectant Not Tested Not Tested necrosis factor (TNF) family, while its counterreceptor on Reactivity on FCM + B cells, CD40, is a member of the TNF receptor/nerve (NGF) receptor family. Interactions between CD40 on B cells and its ligand on activated T cells results INTENDED USE: in activation, proliferation, aggregation, and For Research Use Only. Not for use in diagnostic procedures. immunoglobulin isotype switching. A defect in the gene encoding CD154 is responsible for X-linked REFERENCES: immunoglobulin deficiency with normal or elevated IgM 1) Ishida, T., et al., J. Immunol. 155, 5527-5535 (1995) (X-linked hyper IgM). These patients produce IgM but fail 2) Kooten, C., et al., Eur. J. Immunol. 24, 787-792 (1994) to produce downstream Ig isotypes, suggesting that 3) Fuleihan, R., et al., J. Clin. Invest. 93, 1315-1320 (1994) interaction between CD40 on B cells and its ligand on 4) Nishioka, Y., et al., J. Immunol. 153, 1027-1036 (1994) activated T cells plays a critical role in Ig heavy chain 5) Tsubata, T., et al., Nature 364, 645-648 (1993) switch recombination. The expression of CD154 on 6) Armitage, R. J., et al., Nature 357, 80 (1992) activated T cells is tightly regulated. B cells down-regulate the expression of functional CD154 in activated T cells PROTOCOL: through inhibition of CD154 mRNA expression and Flow cytometric analysis for floating cells release of soluble CD40 that binds CD154. We usually use Fisher tubes or equivalents as reaction tubes for all step described below. SOURCE: This antibody was purified from mouse 1) Wash the cells 3 times with washing buffer [PBS ascites fluid using A agarose. This hybridoma was containing 2% fetal calf serum (FCS) and 0.1% NaN3]. established by fusion of mouse myeloma cell NS-1 with 2) Resuspend the cells with washing buffer (5x106 Balb/c mouse splenocyte immunized with human CD154 cells/mL). transfected 300-19 cell (mouse pre-B cell line). 3) Add 50 L of the cell suspension into each tube, and centrifuge at 500 x g for 1 minute at room temperature o FORMULATION: In 10 mM NaPB (pH 8.0) (20~25 C). Remove supernatant by careful aspiration. 4) Add 10 L of normal goat serum containing 1 mg/mL containing 1% BSA, 0.15 M NaCl and 0.09% NaN3. *Azide may react with copper or lead in plumbing system to normal human IgG and 0.1% NaN3 to the cell pellet after form explosive metal azides. Therefore, always flush plenty of tapping. Mix well and incubate for 5 minutes at room water when disposing materials containing azaide into drain. temperature. 5) Add 20 µL of the FITC labeled CD154 monoclonal STORAGE: This antibody solution is stable for one year antibody (5F3). Mix well and incubate for 30 minutes at from the date of purchase when stored at 4°C. room temperature. 6) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room REACTIVITY: This antibody reacts with human temperature. Remove supernatant by careful aspiration. CD154 on Flow cytometry. 7) Resuspend the cells with 500 L of the washing buffer and analyze by a flow cytometer. APPLICATIONS: Western blotting; Not tested (Positive control for Flow cytometry; transfectant) Immunoprecipitation; Not tested Immunohistochemistry; Not tested Immunocytochemistry; Not tested Flow cytometry; 20 L (ready for use)

Detailed procedure is provided in the following 061211-2.1 PROTOCOL.

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