BD, 557764, BD™ DimerX DimerX I: Recombinant Soluble Dimeric Human CD1d:Ig Fusion Protein

Isotype: Mouse IgG1, λ
Application: ELISA (Routinely Tested), Flow cytometry (Tested During Development)
Concentration: 0.5 mg/ml
RRID: AB_2869093
Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage Store undiluted at 4°C. The human CD1d:Ig fusion protein was expressed together with human β2M in the mouse plasmacytoma cell line, J558L (ATCC TIB-6). The CD1d and β2M polypeptide chains are associated noncovalently as a consequence of their coexpression within J558L cells. The CD1d:Ig fusion protein was purified from tissue culture supernatant by affinity chromatography.The purity of the preparation was confirmed by SDS-PAGE. Additionally, human β2M was added to the reagent. The human CD1d:Ig is tested by ELISA to verify the fusion protein.
Recommended Assay Procedures: Recommended Assay Procedures It is necessary to load the CD1d portions of the dimeric protein with a relevant antigen of interest prior to immunofluorescent staining of NKT cells. CD1d:Ig complexes are effectively loaded by incubation with excess relevant (specific) or irrelevant (control) antigens (see Protocol 1). Antigen-loaded CD1d:Ig may be used for immunofluorescent staining (see Protocol 2). Since applications vary, each investigator must determine dilutions appropriate for individual use . Protocol 1: Antigen Loading of CD1d:Ig Dimeric Protein Several antigen-loading protocols have been described. The method recommended at BD Biosciences Pharmingen involves passive loading of excess antigen in solution with CD1d:Ig protein. We have found that passive loading works particularly well in the case of high-affinity antigens. For lower-affinity antigens, an increase in the molar ratio of antigen to dimer protein may improve loading, as determined by flow cytometric analysis based on results for other BD Pharmingen DimerX products. It is suggested that for each antigen, parameters such as the dose of CD1d:Ig per million cells, molar ratio of antigen to CD1d:Ig, and antigen-loading time be determined empirically by the investigator. Antigen preparation and loading: 1.   The molecular weight (MW) of an antigen of interest will need to be determined. The MW of a-GalCer is 858 daltons. 2.   Mix CD1d:Ig protein with specific or control antigen at 10, 20, or 40 molar (M) excess. The following calculation, using α-GalCer as an example, may be used: Dg = Molecular Weight of antigen: e.g., 858 daltons. DCD1d = Molecular Weight of CD1d:Ig = 250,000 daltons. R = desired excess molar ratio, e.g., 40. Mg = micrograms (µg) antigen of interest. MCD1d = micrograms (µg) CD1d:Ig in the reaction. A typical amount of antigen-loaded CD1d:Ig to use for flow cytometry staining is 0.25 to 4 µg/million cells (test). Mg = MCD1d x R x Dg = 4 µg x 40 x 858 d = 0.55 µg DCD1d 250,000 d Therefore, one would add 0.55 µg of antigen and 4 µg of CD1d:Ig in solution for the optimal antigen loading of CD1d:Ig. 3. Mix antigen and CD1d :Ig together in PBS, pH 7.2, incubate at 37°C overnight.  The antigen-loaded CD1d:Ig can be stored at 4°C for up to 1 week. *NOTE: The rights to α-GalCer are owned by Kirin Brewery. The α-GalCer molecule and its derivatives are covered by US Patent No. 5,936,076. There is no implied license hereunder for the use of α-GalCer. Protocol 2: Immunofluorescent Staining Protocol 1.  Resuspend PBMC's or target cells in FACS staining buffer [e.g., BD Pharmingen™ Stain Buffer with BSA, Cat. no. 554657], at a concentration of approximately 10e6 cells per 50 µl. Add ~1 x 10e6 cells per staining tube (eg, 12 x 75 mm tube, BD Falcon™ Cat. no. 352008). 2.  Prepare antigen-loaded CD1d:Ig protein staining cocktail by mixing 0.25 - 4 µg of antigen-loaded CD1d:Ig protein/test with 0.25 - 4 µg of PE-conjugated A85-1 mAb (anti-mouse IgG1, Cat. No. 550083)/test at a ratio of 1:1 or 1:2 of dimer: A85-1 mAb. Incubate the mixture for 60 minutes at room temperature, protect from exposure to light. 3.  Add 1 -2 µg of purified mouse IgG1 isotype control mAb A111-3 (Cat. No. 553485)/test to the staining cocktail (see Step 2 above). Incubate the staining cocktail for 30 minutes at RT, protect from exposure to light. 4.  Prepare purified polyclonal human IgG at approximately 0.2 mg/ml in phosphate buffered saline (PBS), pH 7.2. 5.  Add 10 µl (2 µg) of human IgG stock per tube to block non-specific binding of DimerX I or antibody reagents to surface Fc receptors. Incubate 10 minutes at room temperature. 6.  Add 50 µl FACS buffer containing the optimal per test amount of the staining cocktail, plus any other cell-surface marker-specific antibodies to be used to each sample. 7.  Wash cells 1x with 2 ml FACS buffer, centrifuge for 5 minutes at 250 x g, and aspirate supernatant. Resuspend in FACS buffer and analyze by flow cytometry. Protocol 3: Alternative: Immunofluorescent Staining Protocol 1.  Resuspend PBMCs or target cells in FACS staining buffer [eg, DPBS, 1% FCS, 0.09% NaN3 or BD Pharmingen™ Stain Buffer (FBS), Cat. No. 554656] at a concentration of approximately 10e6 cells per 50 µl. Add ~1 x 10e6 cells per staining tube (e.g., 12 x 75 mm tube, BD Falcon™ Cat. no. 352008). 2.  Prepare purified polyclonal human IgG at approximately 0.2 mg/ml in phosphate buffered saline (PBS), pH 7.2. 3.  Add 10 µl (2 µg) of human IgG stock per tube to block non-specific binding of DimerX I or antibody reagents to surface Fc receptors. Incubate 10 minutes at room temperature. 4.  Add 0.25 to 4 µg of antigen-loaded CD1d:Ig protein to cell suspension. Incubate 60 minutes at 4°C. 5.  Wash cells 1x with 2 ml FACS buffer, centrifuge for 5 minutes at 250 x g, and discard supernatant. 6.  Again add 10 µl (2 µg) of purified polyclonal human IgG per sample. 7.  Resuspend cells in 100 µl FACS buffer containing appropriately diluted fluorescent secondary reagent. We typically use PE-conjugated A85-1 mAb (anti-mouse IgG1, Cat. No. 550083). Incubate 30 - 60 minutes at 4°C. 8.  Wash cells 1x with 2 ml FACS buffer, centrifuge for 5 minutes at 250 x g, and discard supernatant. 9.  Repeat Steps 7 and 8 for labeling of cell-surface markers with the appropriate fluorochrome-conjugated antibody (avoid antibodies with mouse IgG1 isotypes to reduce background). 10. Resuspend cell pellet in approximately 0.5 ml staining buffer in a tube appropriate for the flow cytometer and analyze.