This H-2Ld:Ig fusion protein has been tested by immunofluorescent staining (≤ 4 µg H-2Ld:Ig/million cells) (see Figures) and flow cytometric analysis of antigen specific T cells to assure specificity and reactivity. It is necessary to load the H-2Ld portions of the dimeric protein with a relevant peptide of interest prior to immunofluorescent staining of T cells. H-2Ld:Ig complexes are effectively loaded by incubation with excess relevant (specific) or irrelevant (control) peptides (see Protocol 1). Peptide-loaded H-2Ld:Ig may be used for immunofluorescent staining (see Protocol 2). Since applications vary, each investigator must determine dilutions appropriate for individual use.
Protocol 1: Peptide Loading of H-2Ld:Ig Dimeric Protein
An alloreactive T-cell clone, 2C, was originally derived by stimulating BALB/c spleen cells with irradiated P815 (H-2Ld) cells. The specificity of 2C is an endogenous peptide, p2Ca, that is derived from 2-oxoglutarate dehydrogenase, presented by Ld. Several related peptides or peptide analogs have been identified. These differ in their MHC restriction and in their affinity for 2C TCR. The peptides QL9 (QLSPFPFDL) or p2Ca (LSPFPFDL) each have high affinity for H-2Ld.
Several peptide-loading protocols have been described. The method used at BD Biosciences Pharmingen involves passive loading of excess peptide in solution with H-2Ld:Ig protein. We have found that passive loading works particularly well in the case of high-affinity peptides. For lower affinity peptides, an increase in the molar ratio of peptide to H-2Ld:Ig may improve loading, as determined by flow cytometric analysis. It is suggested that for each peptide, parameters such as the dose of H-2Ld:Ig per million cells, molar ratio of peptide to H-2Ld:Ig, and peptide loading time be determined empirically by the investigator. While this DimerX product contains β2 Microglobulin, for investigators requiring excess recombinant Human β2 Microglobulin, we recommend BD Biosciences Cat. No. 551089.
Peptide preparation and loading:
1. The molecular weight (MW) of a peptide of interest will need to be determined. A peptide's MW can be estimated by multiplying its number (n) of amino acids (AA) by 130 daltons (d) per amino acid:
MW of peptide (d) = n (AA) x 130 (d/AA)
2. A stock of peptide may be prepared at 20 mg/ml in DMSO. Dilute the peptide solution to 2 mg/ml in sterile DPBS, pH 7.2 for use in the H-2Ld:Ig loading protocol.
3. Mix H-2Ld:Ig protein with specific or control peptide at 40, 160, or 640 molar (M) excess.
The following calculation, using an 8 amino acid peptide (8mer) as an example, may be used:
Dp = Molecular Weight of peptide: e.g., 8 amino acids x 130 = 1,040 daltons.
DLd = Molecular Weight of H-2Ld:Ig = 250,000 daltons.
R = desired excess molar ratio, e.g., 160.
Mp = micrograms (µg) peptide of interest.
MLd = micrograms (µg) H-2Ld:Ig in the reaction. A typical amount of peptide-loaded H-2Ld:Ig to use for flow cytometry staining is 0.25 to 4 µg/million cells (test).
Mp = MLd x R x Dp = 4 µg x 160 x 1,040 d = 2.66 µg Therefore, one would add 2.66 µg of peptide and 4 µg of H-2Ld:Ig
DLd 250,000 d in solution for the optimal peptide loading of H-2Ld:Ig.
4. Mix peptide and H-2Ld:Ig together in PBS, pH 7.2, incubate at 37°C overnight. The peptide-loaded H-2Ld:Ig can be stored at 4°C for up to 1 week.
Protocol 2: Immunofluorescent Staining Protocol
1. Prepare peptide-loaded H-2Ld protein staining cocktail by mixing 0.25 - 4 µg of peptide-loaded H-2Ld 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 RT, protect from exposure to light.
2. Add 0.25 - 4 µg of purified mouse IgG1 isotype control mAb A111-3 (Cat. No. 553485)/test to the staining cocktail (see Step 1 above). Incubate the staining cocktail for 30 minutes at RT, protect from exposure to light.
3. Resuspend mouse cells in FACS staining buffer [e.g., DPBS, 1% FCS, 0.09% NaN3 or BD Pharmingen™ Stain Buffer (FBS), Cat. No. 554656], containing the appropriate amount of Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2 (Cat. no. 553141/553142), at a concentration of approximately 10e6 cells per 50 µl. Incubate 10 minutes at 4°C. Add ~1 x 10e6 cells per staining tube (e.g., 12 x 75 mm tube, BD Falcon™ Cat. No. 352008).
4. Add 50 µl FACS buffer containing the optimal per test amount of the staining cocktail to each sample, plus any other cell-surface marker specific antibodies to be used.
5. Wash cells 2x with 2 ml FACS buffer, centrifuge for 5 minutes at 250 x g, and discard supernatant. Resuspend cell pellet in approximately 0.5 ml staining buffer in a tube appropriate for the flow cytometer.
Protocol 3: Alternative: Immunofluorescent Staining Protocol
1. Resuspend mouse cells in FACS staining buffer [e.g., DPBS, 1% FCS, 0.09% NaN3 or BD Pharmingen™ Stain Buffer (FBS), Cat. No. 554656], containing the appropriate amount of Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2 (Cat. No. 553141/553142), at a concentration of approximately 10e6 cells per 50 µl. Incubate 10 minutes at 4°C. Add ~1 x 10e6 cells per staining tube (e.g., 12 x 75 mm tube, BD Falcon™ Cat. No. 352008).
2. Add 0.25 to 4 µg of peptide-loaded H-2Ld:Ig protein to cell suspension. Incubate 60 minutes at 4°C.
3. Wash cells 1x with 2 ml FACS buffer, centrifuge for 5 minutes at 250 x g, and aspirate supernatant.
4. 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.
5. Wash cells 2x with 2 ml FACS buffer, centrifuge for 5 minutes at 250 x g, and discard supernatant. Resuspend cell pellet in approximately 0.5 ml staining buffer in a tube appropriate for the flow cytometer.
