Tmb elisa development kit

For flow cytometric analysis of cytokine production, coculture cells of day 21 were stimulated in bulk during 6 h with phorbol myristate acetate (PMA; 5 ng/mL) and ionomycin (1 µg/mL) or with K562 cells at an effector to target ratio (E:T) of 1:1, or during 24 h with IL-12 plus IL-18 (both 10 ng/mL) or IL-12, IL-18 and IL-15 (4 ng/mL). The last 4 h of incubation, brefeldin A (BD Golgiplug, BD Biosciences) was added. After harvesting, cells were stained for NK surface markers and subsequently fixed and permeabilized for intracellular staining of IFN-γ and TNF-α. For analysis of cytokine secretion, sorted mature eGFP⁺ NK cells (CD45⁺CD56⁺CD94⁺) from day 21 cultures were stimulated with IL-12 plus IL-18 or IL-12, IL-18 and IL-15 (same concentrations as indicated above). After 24 h, supernatant was collected and analyzed for cytokine secretion with IFN-γ ELISA assay (PeliKine-Tool Set, Sanquin, Amsterdam, The Netherlands) and TNF-α ELISA assay kits (TMB ELISA Development Kit, Peprotech).

For flow cytometric analysis of cytokine production, coculture cells of day 21 were stimulated in bulk during 6 h with phorbol myristate acetate (PMA; 5 ng/mL) and ionomycin (1 µg/mL) or with K562 cells at an effector to target ratio (E:T) of 1:1, or during 24 h with IL-12 plus IL-18 (both 10 ng/mL) or IL-12, IL-18, and IL-15 (4 ng/mL). In the last 4 h of incubation, brefeldin A (BD Golgiplug, BD Biosciences) was added. After harvesting, cells were stained for NK surface markers and subsequently fixed and permeabilized for intracellular staining of IFN-γ and TNF-α. For analysis of cytokine secretion, sorted mature eGFP⁺ NK cells (CD45⁺CD56⁺CD94⁺) from day 21 cultures were stimulated with IL-12 plus IL-18 or IL-12, IL-18, and IL-15 (same concentrations as indicated above). After 24 h, the supernatant was collected and analyzed for cytokine secretion with IFN-γ ELISA assay (PeliKine-Tool Set, Sanquin, Amsterdam, The Netherlands) and TNF-α ELISA assay kits (TMB ELISA Development Kit, Peprotech).

The production of
interleukin 6 (IL-6), tumor necrosis factor alpha
(TNF-α), interleukin 1 beta (IL-1β), and monocyte chemoattractant
protein 1 (MCP-1) by macrophages was measured using enzyme-linked
immunosorbent assay (ELISA). Cell culture supernatants were collected
after 24 h treatments of M0 and M1 THP-1-derived macrophages with
HDPs (0.005–0.5–5 mg/mL) and centrifuged for 10 min,
240g, 4 °C, and stored at −20 °C
until further measurements. DuoSet ELISA Development Systems kit (#DY201;
R&D Systems, Abingdon, UK) was used to quantify the IL-1β
levels in complete cell culture medium supernatants following manufacturer’s
instructions. Standard TMB ELISA Development kits were used to quantify
the remaining cytokines (IL-6 #900-T16, TNF-α #900-T25, and
MCP-1 #900-T31; PeproTech, London, UK) according to the manufacturer’s
protocols. The optical density was determined using a GloMax Discover
Microplate Reader (Promega, Madison, WI, USA) set to 450 nm. Each
sample was measured in duplicate, and quantification was done using
a four-parameter logistic (4-PL) curve fit. Data were presented as
concentration (pg/mL) normalized to DNA content quantified using Quant-iT
PicoGreen assay, as described above.