Cytokine analysis—ELISA

Tumor necrosis factor-alpha, interleukin 6, and interleukin 10 (TNFα, IL6, and IL10, respectively) were quantitated by multiplexed ELISA using the Ella Simple Plex platform from Bio-Techne (Minneapolis, MN) according to manufacturer’s recommendations. Conditioned media from stimulated or unstimulated PBMSC-MSC cocultures treated as described above in 96-well plates were frozen, thawed on the date of assay, and centrifuged at 20,000 × g for 5 min before analysis. Samples were diluted threefold in ELISA kit assay buffer; 60 µL from each sample was added to the dedicated ELISA plate and the plate loaded onto the automated ELISA. Cytokine levels were determined after 70 min. Alternatively, a rapid, no-wash ELISA (Lumit TNFα ELISA, Promega) was performed, which relies on proximity interactions of bound antibody to reconstitute a light-generating enzyme that can produce luminescence signals. Samples were either from frozen, thawed, centrifuged media from mitogen-stimulated assays or on samples taken directly from mitogen-stimulated samples without centrifugation and were assayed according to the manufacturer’s recommendations. For thawed frozen samples, 50 µL each of diluted standards or centrifuged media was added to a 96-well Lumitrac plate followed by the addition of 50 µL of a 2× antibody mix and then 60-min room temperature incubation, per the manufacturer’s recommendations. For freshly harvested samples, 80 µL of diluted standards or resuspended PBMCs was transferred to a 96-well Lumitrac plate with 20 µL of 5× antibody mix, per the manufacturer’s recommendations. After incubation, 25 µL of room temperature equilibrated, diluted detection substrate in buffer was added to each well. After brief mixing, and a 3- to 5-min incubation, the luminescence was read on a luminometer. The concentration of TNFα was determined from the linear standard curve. Suppression of the PBMC signal in the presence of MSCs was calculated as described above.