Human granzyme b elisa development kit

The assays were performed as previously described for IFN-γ [7 (link)]. Briefly, heparinized whole blood (100 μL) was added to flat-bottom microtiter plates and incubated with varying amounts of influenza antigens (HA titer: 10 μg/mL) diluted in RPMI 1640 medium in a final volume of 200 μL/well. The incubations were conducted within 1 h of drawing the blood samples. The supernatants (100 μL) were collected after 48 h of incubation and stored at −80 °C until further use. IFN-γ and granzyme B concentrations were measured using enzyme-linked immunosorbent assays (IFN-γ Assay Kit (ebioscience, San Diego, CA, USA) and a Human Granzyme B ELISA development kit (MABTECH, Nacka Strand, Sweden), according to the manufacturer’s instructions. Phytohemagglutinin (final concentration: 2.5 μg/mL) and medium were used as positive and negative controls, respectively. The amount of IFN-γ released in the negative control wells was < 4 pg/mL in all experiments.
Subjects with a ≥1.5-fold increase in IFN-γ and granzyme B production were considered to have positive reactions because, in our previous study, the fold increase in the IFN-γ production of influenza-infected subjects in response to vaccination was not higher than 1.5 [7 (link)].

Anti-CD16 antibody (clone 3G8) was purchased from BioLegend. BUV395-CD3 (clone UCHT1), PE-Cy™7-CD56 (clone B159), BV421-CD210a (clone 3F9), BV421-IgG2a (clone R35-95), FITC-CD107a (clone H4A3), PE-FasL (clone NOK-1) were purchase from BD Biosciences. Antibodies used for western blot were as follows: mouse anti-β-actin was purchased from Santa Cruz Biotechnology. Rabbit anti-phospho-S6 ribosomal protein (Ser235-Ser236) antibody, rabbit anti-phospho-p70 S6 kinase (Thr389) antibody, rabbit anti-p70 S6 kinase antibody, and mouse anti-phospho-STAT3 (Tyr705) antibody were purchased from Cell Signaling Technology.
Human IL-10 was purchased from Miltenyi Biotec, and human recombinant IL-2 was from R&D System. Cell-Tak™ Cell and Tissue Adhesive was purchased from Corning. Rapamycin and CellTrace Violet were from Invitrogen. Ficoll® Paque Plus, 2-deoxyglucose (2-DG), etomoxir, UK5099, BPTES, oligomycin, FCCP, rotenone, antimycin and concanamycin A were purchased from Sigma-Aldrich. Human IFN-γ ELISA MAX™ Standard Set was from BioLegend. Human Granzyme B ELISA development kit was from Mabtech. Lactate Assay Kit was from Sigma-Aldrich. Fixable viability dye eFluor 780 (FVD) was purchased from eBioscience. 2-NBDG was purchased from Life Technologies. Stattic was from Santa Cruz Biotechnology. SuperSignal West Pico/Dura chemiluminescent substrate was from Pierce.

Supernatants of T cell-tumor cell co-cultures and anti-CD3/CD28 activated T cells were analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA #555142), Tumor necrosis factor alpha (TNFα) (Human TNF ELISA Set, BD OptEIA #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05). Experiments were performed according to the manufacturer´s instructions. Absorbance was measured at λ = 450 nm, taking λ = 570 nm as reference wavelength using the Spark 10 M multimode microplate reader (TECAN). Soluble Fas ligand (FasL) was detected by multiplex cytokine assay (MILLIPLEX Human CD8⁺ T Cell MAGNETIC Premixed 17 Plex Kit, Merck, #HCD8MAG15K17PMX). The assay was performed according to the manufacturer´s instructions and samples were measured using the MAGPIX Luminex instrument (Merck).