5 5 dithiobis 2 nitrobenzoic acid dtmb

96-well flat-bottomed plates were pre-coated overnight at 4°C with 5 µg/ml anti-NKp30 (clone P30-15, Biolegend) and -CD18 (clone IB4) or with mouse IgG1aκ as an isotype control. Wells were blocked with phenol red-free RPMI complete medium then washed three times with PBS. 10⁵ WT or CD56-KO NK92 cells were added per well. Plates containing cells were briefly centrifuged then incubated for 90 min at 37°C 5% CO₂. Following incubation 1% v/v IGEPAL Sigma-Aldrich) was added to maximum release wells and plates were centrifuged at 1000 rpm. Supernatant was transferred and substrate solution containing PBS, HEPES (Gibco), N-α-Cbz-L-lysine thiobenzyl ester hydrochloride (BLT; Sigma-Aldrich) and 5,5’-Dithiobis(2-nitrobenzoic acid) (DTMB; Sigma-Aldrich) was added. The plate was incubated at 37°C 5% CO₂ for 30–60 min and luminescence was read at 415 nm using the BioTek Synergy H4 Hybrid plate reader. % maximum activity was calculated as: (sample absorbance – average background)/(average total release – average background) x 100%.

One hour following the final CPF dosing on PND 4, pups were euthanized by decapitation and blood was collected by cardiac puncture into tubes containing EDTA as an anti-coagulant (Becton-Dickinson, Franklin Lakes, NJ). Blood was diluted 1:25 with phosphate buffer with 0.03% Triton X-100 (Fisher Scientific, Pittsburg, PA), vortexed, and snap frozen for later analysis. Brains were collected and snap frozen for later analysis. For the AChE activity assay, brain tissue was thawed on ice, homogenized in phosphate buffer with 1% Triton X-100, and AChE activity quantified using the standard Ellman Assay [39 (link)] with 5,5′-dithio-bis-2-nitrobenzoic acid (DTMB) and acetylthiocholine iodide (ASChI) as the substrates (Sigma-Aldrich, St. Louis, MO). Tetraisopropyl pyrophosphoramide (Sigma) was included to inhibit pseudocholinesterase. Blood AChE activity was normalized to hemoglobin levels, which were determined using a StanBio Laboratory Stat-Site M hemoglobin meter and test strips (Boerne, TX, USA). Brain AChE activity was normalized to protein concentration as determined using the BCA assay kit (Pierce, Rockford, IL).