Anti mouse cd3 fitc

The monoclonal antibody (mAb) clone 14 M is a mouse anti-mouse IgG1 mAb against B7-H3 generated by the immunization of a B7-H3KO mouse. PE anti-mouse CD8a, PE-cy7 or APCanti-mouse CD4, FITC anti-mouse CD3, PE anti-mouse CD11c and isotype-matched mAbs were purchased from BD Biosciences; and anti-p-STAT3 antibody was purchased from Cell Signaling Technology. Fixation/Permeabilization Kit were purchased from eBioscience. For cell surface staining, cells were directly stained with either IgG control or fluorescence-conjugated antibodies. For intracellular staining, cells were permeabilized and fixed after surface staining, and stained with fluorescence-conjugated antibodies or IgG controls.

For surface staining alone, splenocytes were resuspended at a concentration of 1 × 10⁶ cells/mL in staining buffer (PBS 1x and 1% FBS). Fc receptors were blocked by the addition of unlabeled anti-CD16/32 (Fc block; BD Pharmingen, San Diego, CA, USA). The leukocytes were then stained for 20 min at 4 °C with the optimal dilution of FITC anti-mouse CD3 and APC anti-mouse CD4 antibody (BD, Pharmingen). Cells were washed twice with staining buffer, resuspended in 100 µL, and an equal volume of 2% formalin was added to fix the cells. After that, cells were treated with permeabilization buffer and intracellular IFNγ and IL-7A cytokines were identified with PE anti-mouse IL-17A and PE-Cy7- anti-mouse IFNγ respectively (BD, Pharmingen). The stained cells were analysed with a BDAccuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

Sixty-three mice were randomly divided into nine groups (seven mice in each). Five groups were immunized i.v. with LMΔ-msv, LIΔ-msv, LMΔ-lacZ, LIΔ-lacZ, and NS. The other four groups were prime-boost immunized i.v. with LMΔ-msv → LMΔ-msv, LMΔ-msv → LIΔ-msv, LIΔ-msv → LMΔ-msv, and LIΔ-msv → LIΔ-msv, respectively. Boost immunization was carried out 40 days after prime immunization. Immunization doses were 0.1-fold of the 50% lethal doses of each strain. Splenocytes were harvested from the immunized mice 9 days after the last immunization. Isolated splenocytes were stimulated with the peptide pool listed in Table 2 at the final concentration of 1 μg/mL prepared in 10 μg/mL Golgi stop (BD PharMingenTM, USA) or no stimulant in 96-well plates at 37°C for 5 h, and then processed for flow cytometry analysis as described previously (16 (link)). After centrifuging for 5 min at 1, 300 × g, the cells were then washed and co-stained with FITC-Anti-Mouse CD3 (BD, USA), PerCP-Anti-Mouse CD4 (BD, USA), and APC-CY7-Anti-Mouse CD8 (BD, USA) at 4°C for 30 min. The surface-stained cells were washed and permeabilized using Cytofix/Cytoperm kit (BD, USA). The cells were then washed and stained for 45 min on 4°C for PE-Anti-Mouse IFN-γ (Biolegend, USA), PE-Cy7-Anti-Mouse TNF-α (Biolegend, USA), and APC-Anti-Mouse IL-2 (Biolegend, USA). The stained cells were analyzed using flojow 10.0.