Igg2a

OVA-specific IgE, IgG₁, and IgG_2a antibody titers in sera were determined by ELISA (Becton Dickinson Biosciences, San Jose, CA, USA) as described previously [13 (link)]. In brief, 96-well microtiter plates were coated with OVA (10 μg/mL) in NaHCO₃ buffer (pH 9.6) at 4°C overnight, and then treated with mouse sera followed by biotin-conjugated anti-mouse IgE, IgG₁, or IgG_2a (PharMingen, San Diego, CA) for 1 h at 37°C. Then, avidin-horseradish peroxidase (1 : 5000, Pierce Biotechnology, Rockford, IL, USA) was added to each well for another 30 min at room temperature. Finally, the reaction was developed by 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich, St. Louis, MO) and the absorbance was determined at 405 nm. The levels of antibody were compared with OVA-specific IgE, IgG₁ and IgG_2a standard serum with predetermined concentrations (immunoglobulin concentrations: IgE = 1.5 μg/mL, IgG₁ = 32.5 μg/mL IgG_2a = 15.2 μg/mL). The concentration of standard serum was arbitrarily assigned as 1 ELISA unit (1 EU).

The expression of isolated MenSCs surface markers was evaluated using fluorescence-activated cell sorting (FACS). Briefly, 5 × 10⁵ cells were collected and washed twice with stain buffer (BD Biosciences, San Jose, CA, USA). MenSCs were incubated in the dark for 20 min with the following primary antibodies: PE-conjugated CD29, CD34, CD45, CD73, CD90, CD105, CD117, and HLA-DR (Becton Dickinson, Franklin Lakes, NJ, USA). The stained cells were washed twice with stain buffer, resuspended in 500 μl of stain buffer and then analyzed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). IgG1 (Becton Dickinson, Franklin Lakes, NJ, USA) was used as an isotype control for the anti-CD29, anti-CD34, anti-CD45, anti-CD73, anti-CD90, anti-CD105, and anti-CD117 antibodies. IgG2a (Becton Dickinson) was used as the isotype control for the anti-HLA-DR antibody. The results were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

For the immunophenotyping of blood leukocytes [25 (link)], separated leukocytes were incubated with mouse monoclonal antibodies to the surface antigens CD4, WC1, and MHC-II and the cell adhesion molecules CD11a, CD11b, and CD18 diluted in MIF buffer for 15 min at 4°C. After incubation, cells were washed 3 times with MIF buffer by centrifugation at 300× g for 3 min and discarded the supernatant. Unlabeled primary antibodies were detected using fluorochrome-labeled secondary antibodies, and labeled cells were then analyzed by flow cytometry. A Becton Dickinson FACSCalibur equipped with Cell Quest software (FACSCalibur; Becton Dickinson Biosciences, San Jose, California, USA) was used to collect the data. At least 100,000 cells were collected and analyzed with the software FlowJo version 10 (Flowjo LLC, USA). Negative isotype controls for mouse IgG1, IgG2a, IgG2b (Becton Dickinson), and IgM (Beckman Coulter, CA, USA). were included as part of the study.

The expression levels of MenSC surface markers were detected by fluorescence-activated cell sorting (FACS). The collected MenSCs (2 × 10⁷) were washed with staining buffer (Becton Dickinson, Biosciences, San Jose, USA) and incubated for 1 h in diluted antibodies, including CD29, CD34, CD45, CD73, CD90, CD105, CD117 and human leukocyte antigen-DR (HLA-DR) (Becton Dickinson, Franklin Lakes, NJ, USA). Isotype antibodies IgG1 and IgG2a (Becton Dickinson) were applied as negative controls. An FC500 flow cytometer (Beckman Coulter, Pasadena, USA) and FlowJo software (Tree Star, Inc., Ashland, OR, USA) were applied for analysis.
A human mesenchymal stem cell osteogenic differentiation medium kit, chondrogenic differentiation medium kit, and adipogenic differentiation medium kit (Cyagen Biosciences, USA) were used to detect the differentiation potential of MenSCs.

Prior to staining, cells were pelleted and washed in phosphate buffered saline containing 0.02% sodium azide, 1% bovine serum albumin, and 1% heat inactivated normal human serum.
Monocytes and moDC were stained for HLA-DR (clone B8.11.2, IgG2b), HLA-A2 and CD14 (both from BD Biosciences), CD206/“mannose receptor” (MR, clone D547.3, IgG1 [30 (link)]), CD209/DC-SIGN (R&D, IgG2a), and CD89 (clone 2D11, IgG1 [31 (link)]). Staining was visualized with secondary antibodies, that is, goat anti-mouse Ig-F(ab)2-APC or goat anti-mouse Ig-F(ab)2-PE (both from Dako, Glostrup, Denmark). Mouse isotypes controls used were IgG1, IgG2a, and IgG2b (all from BD Biosciences). Staining was visualized by flow cytometry on a FACSCalibur equipped with CellQuest software (both from BD). Viable cells were gated based firstly on FSC/SSC and secondly using Annexin-V/PI staining kit (Molecular probes, Invitrogen, USA) to distinguish between viable and dead or apoptotic cells. In all experiments only viable cells were selected.

Specific IgG1 and IgG2a against R. rickettsii antigens were measured in the serum of mice receiving DC adoptive transfer under different conditions by an in-house ELISA assay. Briefly, 96-well plates were coated with HKRr (equivalent to 10⁶ bacteria/well) diluted in 0.1 M sodium carbonate buffer (pH 9.5) and maintained overnight at 4°C, followed by blocking with PBS containing 1% heat-inactivated FBS for 1 h at room temperature. Serum samples (1:100 dilution) were added and incubated for 2 h. Peroxidase-labeled anti-IgG1 (Invitrogen, Waltham, MA, United States) and -IgG2a (BD Biosciences) detection antibodies (1:1,000 dilution) were added for 1 h and the reaction was developed by addition of the TMB substrate reagent set (BD Biosciences). The reaction was stopped by addition of phosphoric acid [(H₃PO₄)1 M] and the absorbance at 450 nm was determined and used to qualitatively estimate the amount of each antibody. The blank for each reaction consisted of the same dilutions of serum from mice injected with DCs only.

IPV-specific antibodies were determined by ELISA. One hundred microliters of a 1:200 dilution of each IPV strain were absorbed overnight at 4 °C to ELISA plates in 0.1 M sodium hydrogen carbonate buffer, pH 9.6. Wells were blocked with 1% BSA/PBS and 100 µL of serum diluted in 1% BSA/PBS (1:1000 for total IgG and IgG1 and antibodies and 1:200 for IgM, IgG2a, IgG2b and IgG3) were added and incubated for 2 h at RT. For murine studies, after washing, HRP-conjugated anti-mouse IgM (BD Bioscience), IgG (Millipore), IgG1 (BD Bioscience), IgG2a (BD Bioscience), IgG2b (BD Bioscience) and IgG3 (BD Bioscience) were added and incubated for 1 h at RT. For rat studies, HRP-Goat anti-Rat IgG (Chemicon, Cat. No. AP136P) was added and incubated for 1 h at RT. After a final wash, plates were incubated with 100 µL of freshly prepared TMB substrate for 10 min and then the reaction stopped by 1 M phosphoric acid and the optical density measured at 450 nm (OD 450 nm) using VersaMax ELISA microplate reader (Molecular Devices) and data analyzed by SoftMax Pro Software.