Anti mouse 1 a 1 e antibody

A T cell hybridoma assay was performed to demonstrate blocking of 52.13 activation in the presence of its epitope. After harvesting and preparing cell suspension from spleen, splenocytes (5 x 10⁵/well) were treated with 4-fold serial dilutions of purified anti-mouse I-A/I-E antibody (Biolegend, clone M5/114.15.2) starting at 2000ng/ml in the presence of 10-fold serial dilutions of peptide 36 (from 100uM to 0.1uM), 0.5ul/well of B. thetaiotaomicron VIP 5482 or no antigen, and incubated for 30 minutes at 4°C. Subsequently, 52.13 hybridomas (5 x 10⁴) were added and the plate was incubated at tissue culture conditions for 22–24 hours. Assay was completed as described above for screening of reactivity to Bacteroides species and transposon mutants.

To evaluate DC maturation in vitro, bone marrow cells were obtained from BALB/c mice and cultured in complete 1640 medium, supplemented with mouse recombinant macrophage colony stimulating factor (GMCSF, 20 ng/ml, Cat# 576304, Biolegend) and IL-4 (10 ng/ml, Cat# 574304, Biolegend) for 6 days to generate bone marrow-derived DCs. Then, IR-780 treated CT26 cancer cells were co-cultured with the DCs for 24 h. Next, the cells were collected and stained with APC anti-mouse CD11c Antibody (Cat# 117310, Biolegend), Brilliant Violet 421™ FITC anti-mouse CD80 Antibody (Cat# 104706, Biolegend), PE anti-mouse CD86 Antibody (Cat# 105008, Biolegend), or anti-mouse I-A/I-E Antibody (Cat# 107632, Biolegend), and analyzed by flow cytometry.

T cells in untreated, female, 6–8 weeks BALB/c mouse spleen were obtained by a mouse CD3 T-cell enrichment kit (Stemcell). Then, T cells were labeled with fluorescence using a CFSE-cell labeling kit (Abcam) to detect T-cell proliferation. BMDCs, primary NECs (obtained from approximately 20 mice), or NEC cell lines were treated with CCD/RBD-HR (10 or 20/1 μg/mL) for 24 h at 37 °C. These treated and untreated BMDCs or NECs (2 × 10⁵ cells/mL) were then coincubated with CFSE-labeled T cells (1 × 10⁶ cells/mL) in a 37 °C incubator for 48 h. Proliferating T cells, activated T cells, and antigen-experienced T cells were then detected by FCM. In the experiment of coculturing NECs and T cells, the effect of MHC II expressed on NECs on CD4⁺ T cells was investigated with a neutralizing anti-mouse I-A/I-E antibody (BioLegend, 2.5 μg/mL). Rat IgG2b, κ (BioLegend) was used as an isotype control.