Apotox glo

Spontaneous apoptosis of mature Trem1^+/+ versus Trem1^−/− neutrophils was assessed at 5 days post differentiation in SCF-containing medium and 8 h, 24 h, and 48 h later. Immediately upon removal from plates, neutrophils were washed in cold PBS and AnnexinV Binding Buffer and stained with FITC-conjugated AnnexinV (BD Pharmingen) according to the manufacturer's instructions. DAPI (Sigma-Aldrich) was added immediately prior to aquisition by flow cytometry. Only AnnexinV and DAPI double-positive cells (AnnexinV⁺ DAPI⁺) were considered in the analysis of apoptotic cells. For determination of TREM-1-mediated effects on apoptosis induction, 5 days differentiated neutrophils were stimulated in vitro in 96-well U-bottom plates at 2×10⁵ cells/well in complete RPMI medium lacking SCF in the presence of 10 µg/ml plate-bound anti-TREM-1 mAb (MAB1187; R&D) or a respective isotype control (RTK2758, Biolegend) for 24 h. Since the relative stickiness of neutrophils activated by plate-bound anti-TREM-1 did not allow for removal from the plates and FACS-based analysis of AnnexinV⁺ DAPI⁺ cells without causing a substantial bias by the selective analysis of non-adherent cells, Caspase 3/7 activity was determined by the ApoTox-Glo assay (Promega) according to manufacturer's instructions.

The ROS assay was conducted in HT22 cells as described previously75 . The cells were cultured in 96-well plates. After 24 h of incubation at 37 °C in a humidified atmosphere of 5% CO₂, the cells were treated with fresh medium containing Aβ_1-42 (5 μM), with or without VA at three different concentrations (50, 100 and 200 μM), and the cells were incubated for an additional 24 h. The control cells received only DMEM. Following this, a 600-μM solution of DCFDA (20, 70-dichlorofluorescein diacetate) dissolved in DMSO/PBS was then added to each well, and the cells were incubated for 30 min. The plates were then read on an ApoTox-Glo (Promega) instrument at 488/530 nm.

The total cholesterol and free cholesterol levels in brain homogenates were quantified using an assay kit (ab65390, Abcam) according to the manufacturer's approved guidelines.
The glutathione (GSH) levels and GSH/oxidized GSH (GSSG) ratio were measured with a fluorometric GSH assay kit (BioVision Inc., Milpitas, CA, USA; cat. no. K264–100), according to the company's product protocol.
ROS levels were examined as previously described [52 (link)]. Briefly, the animal's brain homogenates were diluted in ice-cold Locke's buffer (1 : 20) to attain a final concentration solution of 5.0 mg/tissue/ml. To attain the fluorescence, 1 ml solution (Locke's buffer, pH 7.4, and 0.2 ml brain homogenate) was incubated with 10 ml of 5 mM dichlorodihydrofluorescein diacetate (DCFH-DA) (Santa Cruz Biotechnology, CAS #4091-99-0) for 30 min resulting in the oxidation of DCFH-DA into fluorescent product dichlorofluorescein (DCF).
The fluorescence intensities of assays were measured (cholesterol: Ex/Em = 535/587nm, GSH: Ex/Em = 340/420nm, and ROS: Ex/Em = 484/530nm) with a 96-well fluorescence microplate reader ApoTox-Glo™ (Triplex Assay, Promega, Madison, WI, USA).