The HER-2/neu peptide antigen was co-administered subcutaneously with either PBS, soluble Hp91, or NP-encapsulated Hp91 on the right flank. Spleens were collected 8 days after the final immunization. Single cell suspensions of splenocytes were prepared by mechanical disruption and separation through a 70-μm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Red blood cells were lysed using ammonium chloride buffer (Roche Diagnostics, Indianapolis, IN, USA) and the splenocytes were subsequently resuspended in complete medium (RPMI 1640 with 10% FBS, L-glutamine, penicillin, streptomycin, and HEPES) supplemented with 20 U/mL of recombinant mouse interleukin (IL)-2) (R&D Systems, Minneapolis, MN, USA) and 10 μg/mL of PDSLRDLSVF peptide for expansion. Splenocytes were expanded for 5 days prior to use in ELISPOT experiments.
Recombinant mouse interleukin il 2
Manufactured by R&D Systems
Sourced in United States
Recombinant mouse interleukin (IL)-2 is a cytokine that plays a crucial role in the activation and proliferation of T cells. It is a protein produced using recombinant DNA technology and is derived from the mouse species.
Automatically generated - may contain errors
Lab products found in correlation
70 μm nylon cell strainer, by BD (1 mentions)
Ammonium chloride buffer, by Roche (1 mentions)
Ly364947, by Merck Group (1 mentions)
Recombinant mouse il 2, by Thermo Fisher Scientific (1 mentions)
Recombinant human tgf β1, by R&D Systems (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd3 cd28 coated beads, by Thermo Fisher Scientific (1 mentions)
2 protocols using recombinant mouse interleukin il 2
1
HER-2/neu Peptide Antigen Immunization
2
CD4+ T Cell Activation by Hepatocyte Supernatants
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CD4 + CD25 -T cells were cultured in different media. Hepa1-6, scramble, and shRNA-TGF-β1-Hepa1-6 cells (2 × 10 6 ) were plated into T25 flasks with complete medium for 2 days, and the supernatants were then replaced with fresh AIMV medium. After a further 3 days, the supernatants were collected and diluted with fresh AIMV medium (1:3) and were called Hepa1-6, scramble, and shRNA-TGF-β1-Hepa1-6 supernatants, respectively. These supernatants were added to 30 U/ml recombinant mouse interleukin (IL)-2 (R&D Systems, Minnesota, USA), 10 mM HEPES buffer (Hyclone), and 0.05 mM 2-ME (Gibco). Isolated CD4 + CD25 -T cells (5 × 10 5 /well) in 48-well plates were cultured in either complete T cell medium (AIMV, 30 U/ml recombinant mouse IL-2, 10 mM HEPES buffer, 0.05 mM 2-ME), or Hepa1-6, scramble, or shRNA-TGF-β1-Hepa1-6 supernatant for 3 days in the presence of anti-CD3/CD28-coated beads (5:1, Invitrogen).
Recombinant human TGF-β1 (1 ng/mL, R&D Systems) and TGF-β1 receptor kinase inhibitor LY-364947 (3 µM, Merck, Germany) were added to some cultures and T cell proliferation was measured using the carboxyfluorescein succinimidyl ester (CFSE) method, according to the manufacturer's instructions (Invitrogen).
Recombinant human TGF-β1 (1 ng/mL, R&D Systems) and TGF-β1 receptor kinase inhibitor LY-364947 (3 µM, Merck, Germany) were added to some cultures and T cell proliferation was measured using the carboxyfluorescein succinimidyl ester (CFSE) method, according to the manufacturer's instructions (Invitrogen).
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