Synergy neo2 multi mode reader

β-hematin crystal growth inhibition was assessed under physiological conditions in the presence of lipid catalyst using a 96-well plate assay previously described.^{[69 (link)]} Briefly, a mixture of 10 μL of 2 mM hemin (Sigma-Aldrich, St. Louis, MO) dissolved in 0.1 M NaOH, 180 μL of propionate buffer at pH 5.2 and 10 μL of previously sonicated phosphatidylcholine (Sigma-Aldrich, St. Louis, MO) suspension (10 mg/mL) were incubated with different concentrations of the test compound at 37 °C with gentle shaking for 16 h. Samples treated with DMSO and CQ were used as controls. The reaction was stopped by the addition of 100 μL of a solution of SDS 7.5% (wt/vol) dissolved in 0.1M bicarbonate buffer (pH 9.1). The well contents were gently mixed and incubated at room temperature for 10 min. A 50 μL of aliquot from each well was transferred to a second plate pre-loaded with 200 μL/well of SDS solution (2.5% wt/vol in 0.1 M bicarbonate buffer), and the absorbance at 405 nm was read using Synergy Neo2 multi-mode reader (BioTek). Free hemin in solution was estimated using a linear calibration curve measured for each assay (hemin concentrations ranging from 0 to 16 μM freshly prepared in 2.5% SDS-0.1 M bicarbonate buffer pH 9.1). Images of crystals were taken before stopping the reaction with SDS using a Nikon Eclipse TE200 inverted microscope.

Protein was collected from murine small intestinal organoids at days 4-5 that had been starved in serum-free DMEM media (Thermo Fisher) for at least 4 hours, treated for 3 hours with inhibitors against REELIN receptors (purified NA/LE Hamster anti-rat CD29 (BD Biosciences) (20 μ/ml), recombinant mouse LRPAP protein (R&D systems) (200 ng/ml)) and stimulated with recombinant REELIN (mouse recombinant REELIN (R&D) (10 nM)) for 10-20 minutes as indicated. After stimulation organoids were washed in PBS, dissociated with the Gentle Cell Dissociation Reagent (Stemcell Technologies), washed twice in PBS with 0.1% BSA and lysed in ~50 μl RIPA Lysis and Extraction Buffer (Thermo Fisher) with added PhosSTOP™ (Roche) and cOmplete Protease Inhibitor Cocktail (Roche). After lysis, samples were spun down at 4°C and 16000g for 15 minutes and the supernatant was transferred to a new tube. We used the Pierce™ BCA Protein Assay Kit (Thermo Fisher) according to manufacturer’s instructions to measure protein amount at 562 nm on a Synergy Neo2 Multi-Mode Reader (BioTek) relative to a BSA standard curve. 10-15 μg of total protein were loaded for all experiments.