The stimulation index (SI) was measured to detect the splenic immune response as described before (Yang et al., 2017 (link)). Total spleen cells were collected and separated into single cells by cell strainer. Subsequently, the cells were aliquoted into 96-well plates (1 × 106 cells/well) and cultured in DMEM containing 10% FBS (HyClone). Then, 5 μg of OmpAVac was added, followed by co-incubation for 36 h to stimulate the spleen cells. Cells treated with 10 mM phytohemagglutinin-A (PHA) (Gibco) and unstimulated cells served as positive and negative controls, respectively. Cell proliferation was determined using cellular incorporation of 5-bromo-2-deoxyuridine (BrdU, Roche, Germany), followed by absorbance measurements (Ab) at 450 nm. The SI was calculated by dividing the Ab of stimulated cells by the Ab of unstimulated cells. Additionally, the supernatant was collected to measure the concentrations of IL-4, INF-gamma, and IL-17A via ELISA according to the manufacturer's instructions (R&D Systems).
Phytohemagglutinin a pha
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phytohemagglutinin-A (PHA) is a lectin protein derived from the red kidney bean (Phaseolus vulgaris). It functions as a potent mitogen, stimulating the proliferation of T lymphocytes in vitro.
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3 protocols using phytohemagglutinin a pha
1
Splenic Immune Response Assay
2
Lymphocyte Proliferation and Cytokine Profiling
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Lymphocyte proliferation was determined using the cellular incorporation of 5-bromo-2-deoxyuridine (BrdU, Roche, Germany) and measured by absorbance at 450 nm30 (link), and the stimulation index (SI) was calculated by dividing the absorbance of stimulated cells by absorbance of unstimulated cells. The lymphocyte proliferation rates and the phenotype of the proliferative cells was evaluated by using the intracellular fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) followed by Flow cytometric analysis31 . APC/Cy7 anti-mouse CD3 Antibody (17A2, Biolegend), PerCP/Cy5.5 anti-mouse CD4 Antibody (RM4-4, Biolegend), APC anti-mouse CD8a Antibody (53−6.7, Biolegend) and PE anti-mouse CD19 Antibody (1D3/CD19, Biolegend) were used for cells staining. Cells treated with 10 mM phytohemagglutinin-A (PHA) (Gibco, USA) or not served as positive and negative controls, respectively. The levels of IFN-γ, IL-4 and IL-17 were measured in 72 hours cultures by examining cell-free culture supernatant fluid using a Mouse Quantikine ELISA kit for IFN-γ, IL-4 or IL-17 (R&D Systems, USA), respectively.
3
Immune Response Evaluation Protocol
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Seven days after the last immunization, the mice were sacrificed by cervical dislocation under anesthetic with pentobarbital and their spleens were taken out under sterile conditions. Single-cell suspension of spleen was prepared according to standard procedure. For proliferative assay, spleen cells grown in a 96-well plate at a concentration of 4 × 10 5 living cells/200 μl per well. We added 30 μM/well of 5-bromo-2-deoxyuridine (BrdU, Roche, Germany) for measurement of lymphocyte proliferation. 10 mM phytohemagglutinin-A (PHA) (Gibco, USA) and PBS was served as positive and negative controls, recombinant protein OmpA and Pal, were added to the plates (10 μg/ml). Spleen cells were incubated at 37.0 °C with 5% CO 2 for 96 h. Dyed incorporation was determined by measuring absorbance at 450 nm [25] (link). The stimulation index (SI) was calculated by dividing the absorbance of the stimulating cells by the absorbance of the unstimulating cells (SI = OD 450 of stimulated cells/OD 450 of unstimulated cells). Then the supernatant of spleen cells was collected and used for cytokines detection. The levels of INF-γ, IL-4 and IL-17A in supernatant were measured by using ELISA kit (Dakewe Biotech Co, China).
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