Sigmafast p nitrophenyl phosphate tablets

The previously-described PIASPAC protocol was applied to assay the relative ALP activity in OBs and UC-MSCs with some modifications [40 (link),41 (link)]. Viable cells were seeded on the cell assay platform as droplets (OB: 1.4 × 10⁴ cells/cm², UC-MSC: 2.9 × 10⁴ cells/cm²). After 7 days of culture, SIGMAFAST™ p-nitrophenyl phosphate tablets (N1891, SIGMA-ALDRICH, St. Louis, MO, USA) dissolved in water were added to the medium following the manufacturer’s protocol and incubated for 30 min. The absorbance of the supernatant was measured by an absorption plate reader (iMark™ Microplate Absorbance Reader) at 405 nm. Significant differences in the data between two conditions were determined by Student’s t-test.

For the immune sera set, antibodies against the recombinant NTS-DBL1α were measured by ELISA as previously described [26 (link)]. Maxisorp plates (Nunc, Rosklid) were coated overnight at 4 °C with 1 μg of the recombinant antigen dissolved in 15 mM Na₂CO₃ and 35 mM NaHCO₃ (pH 9.6). Plates were washed three times with PBS containing 0.1 % Tween 20 (PBST) and then blocked with 1 % bovine serum albumin (BSA) in PBST for 1 h at room temperature and subsequently washed three times with PBST. All samples were titrated by six serial dilutions in PBS to determine concentration dependency, and they were incubated in triplicate for 1 h at room temperature. Plates were washed three times with PBST, then bound IgG was measured by incubation for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-human IgG (Sigma) diluted 1:1000 in PBS. Plates were washed three times with PBST and developed with SigmaFast p-nitrophenyl phosphate tablets (Sigma) for 15 min. The optical density (OD) was measured at 405 nm in an ELISA reader (Multiskan™ GO Microplate Spetrophotometer, Thermo Scientific). A control pool of six non-malaria exposed Swedish donors was included, as well as control wells without serum (background). Seropositivity was determined based on the 1:500 dilutions as the mean OD₄₀₅ plus two standard deviations of the Swedish control pool.

We used serial plasma dilutions (10%, 5%, 2.5%, 1.25%, and 0.75% in BBS) and pooled plasma of mice belonging to the same experimental group, incubating the samples for 15 min on mannan- (10 µg/mL) or acBSA-coated (25 µg/mL) plates prepared according to a procedure reported previously.^{18 (link)} Plates were washed and incubated for 1 h 30 min at RT with a polyclonal anti-human-C3c antibody (Dako, A0062) diluted to 2.4 µg/mL in wash buffer. After washing, plates were incubated with an alkaline phosphatase-labeled goat anti-rabbit IgG antibody (Sigma A-3812) diluted to 1 µg/mL in wash buffer for 1 h 30 min at RT. After washing, the assay was developed by adding 100 µL of substrate solution (Sigma Fast p-Nitrophenyl Phosphate Tablets, Sigma). The absorbance at 405 nm was then measured using the Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).